Formulation of adenovirus for gene therapy

ABSTRACT

The present invention addresses the need to improve the long-term storage stability (i.e. infectivity) of vector formulations. In particular, it has been demonstrated that for adenovirus, the use of bulking agents, cryoprotectants and lyoprotectants imparts desired properties that allow both lyophilized and liquid adenovirus formulations to be stored at 4° C. for up to 6 months and retain an infectivity between 60-100% of the starting infectivity.

[0001] The present application claims priority to the contents of U.S.Provisional Patent Application Ser. No. 60/108,606, filed Nov. 16, 1998and U.S. Provisional Patent Application Ser. No. 60/133,116, filed May7, 1999. The entire text of the above-referenced disclosure isspecifically incorporated by reference herein without disclaimer.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields ofmolecular biology, virus production and gene therapy. More particularly,it concerns methods for the formulation of highly purified lyophilizedand liquid adenovirus particles stable for long-term storage. Animportant embodiment of the present invention is the use of suchlong-term storage virus preparations for gene therapy treatments ofviral disease, genetic disease and malignancies.

[0004] 2. Description of Related Art

[0005] Viruses are highly efficient at nucleic acid delivery to specificcell types, while often avoiding detection by the infected hosts immunesystem. These features make certain viruses attractive candidates asgene-delivery vehicles for use in gene therapies (Robbins andGhivizzani; 1998; Cristiano et al., 1998). Retrovirus, adenovirus,adeno-associated virus (AAV), and herpes simplex virus are examples ofcommonly used viruses in gene therapies. Each of the aforementionedviruses has its advantages and limitations, and must therefore beselected according to suitability of a given gene therapy (Robbins andGhivizzani; 1998).

[0006] A variety of cancer and genetic diseases currently are beingaddressed by gene therapy. Cardiovascular disease (Morishita et al.,1998), colorectal cancer (Fujiwara and Tanaka, 1998), lung cancer (Rothet al., 1998), brain tumors (Badie et al., 1998), and thyroid carcinoma(Braiden et al., 1998) are examples of gene therapy treatments currentlyunder investigation. Further, the use of viral vectors in combinationwith other cancer treatments also is an avenue of current research(Jounaidi et al., 1998).

[0007] Viral particles must maintain their structural integrity to beinfectious and biologically active. The structural integrity of a viralvector often is compromised during the formulation process, thusprecluding its use as a gene therapy vector. Adenoviruses for genetherapy traditionally have been formulated in buffers containing 10%glycerol. Formulated adenovirus is stored at <−60° C. to ensure goodvirus stability during storage. This ultra-low temperature storage notonly is very expensive, but creates significant inconvenience forstorage, transportation and clinic use. There is an urgent need todevelop new formulation for adenovirus that can be stored atrefrigerated condition.

[0008] Lyophilization has been used widely to improve the stability ofvarious viral vaccine and recombinant protein products. It is expectedthat the long-term storage stability of adenovirus can be improved byreducing the residual water content (moisture) in the formulated productthrough lyophilization. However, there have not been reported studies onthe lyophilization of live adenovirus for gene therapy.

[0009] Generally it is assumed that adenovirus will not maintain itsinfectivity when stored at refrigerated condition in a liquid form forextended period of time. As a result, there are no reported studies onformulating and storing adenovirus at refrigerated condition in a liquidform. Thus, there remains a need for long-term storage stableformulations of viral preparations.

SUMMARY OF THE INVENTION

[0010] The present invention addresses the need for improved, storagestable viral formulations, and methods for the production thereof, foruse in gene therapy. In particular embodiments, a pharmaceuticaladenovirus composition comprising adenovirus particles andpharmaceutical excipients, the excipients including a bulking agent andone or more protectants, wherein the excipients are included in amountseffective to provide an adenovirus composition that is storage stable.In preferred embodiments, the adenovirus composition has an infectivityof between 60 and 100% of the starting infectivity, and a residualmoisture of less than about 5%, when stored for six months at 4°centigrade.

[0011] In one embodiment, the adenovirus composition is a freeze driedcomposition. In particular embodiments, the bulking agent in the freezedried adenovirus composition forms crystals during freezing, wherein thebulking agent is mannitol, inositol, lactitol, xylitol, isomaltol,sorbitol, gelatin, agar, pectin, casein, dried skim milk, dried wholemilk, silcate, carboxypolymethylene, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methhylcellulose ormethylcellulose.

[0012] In certain embodiments, the bulking agent in the freeze driedadenovirus composition is mannitol. In other embodiments the compositionis further defined as an aqueous composition comprising mannitol in aconcentration of from about 1% to about 10% (w/v). In anotherembodiment, the aqueous composition comprises the mannitol in aconcentration of from about 3% to 8%. In a preferred embodiment, theaqueous composition comprises mannitol in a concentration of from about5% to 7%.

[0013] In certain embodiments, the freeze dried composition is preparedfrom an aqueous composition comprising a bulking agent in aconcentration of from about 1% to 10% (w/v). In other embodiments thefreeze dried composition is prepared from an aqueous compositioncomprising a bulking agent in a concentration of from about 3% to 8%. Inyet other embodiments, the freeze dried composition is prepared from anaqueous composition comprising a bulking agent in a concentration offrom about 5% to 7%.

[0014] In particular embodiments, pharmaceutical excipients serve as aprotectants. In one embodiment, the protectant is further defined as acryoprotectant. In certain embodiments, the cryoprotectant is anon-reducing sugar. In particularly defined embodiments the non-reducingsugar is sucrose or trehalose. In preferred embodiments the non-reducingsugar is sucrose.

[0015] In certain embodiments, the composition is further defined as anaqueous composition comprising a non-reducing sugar in a concentrationof from about 2% to about 10% (w/v). In other embodiments, the aqueouscomposition comprises the sugar in a concentration of from about 4% to8%. In still other embodiments, the aqueous composition comprises thesugar in a concentration of from about 5% to 6%.

[0016] In one embodiment, the freeze dried composition is prepared froman aqueous composition comprising a non-reducing sugar in aconcentration of from about 2% to 10% (w/v). In other embodiments, thefreeze dried composition is prepared from an aqueous compositioncomprising a non-reducing sugar in a concentration of from about 4% to8%. In yet other embodiments, the freeze dried composition is preparedfrom an aqueous composition comprising a non-reducing sugar in aconcentration of from about 5% to 6%.

[0017] In another embodiment, the cryoprotectant is niacinamide,creatinine, monosodium glutamate, dimethyl sulfoxide or sweet wheysolids.

[0018] In certain embodiments, the protectant includes a lyoprotectant,wherein the lyoprotectant is human serum albumin, bovine serum albumin,PEG, glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80. In a preferred embodiment, the lyoprotectant is human serumalbumin.

[0019] In certain embodiments, the composition is further defined as anaqueous composition comprising the lyoprotectant in a concentration offrom about 0.5% to about 5% (w/v). In another embodiment, the aqueouscomposition comprises the lyoprotectant in a concentration of from about1% to about 4%. In still another embodiment, the aqueous compositioncomprises the lyoprotectant in a concentration of from about 1% to about3%.

[0020] In particular embodiments, the freeze dried composition isprepared from an aqueous composition comprising a lyoprotectant in aconcentration of from about 0.5% to 5% (w/v). In other embodiments, thefreeze dried composition is-prepared from an aqueous compositioncomprising a lyoprotectant in a concentration of from about 1% to 4%. Inanother embodiment, the freeze dried composition is prepared from anaqueous composition comprising a lyoprotectant in a concentration offrom about 1% to 3%.

[0021] In one embodiment, pharmaceutical excipients defined asprotectants, comprise both a lyoprotectant and a cryoprotectant.

[0022] Also contemplated in the present invention is an aqueouspharmaceutical adenovirus composition comprising a polyol in an amounteffective to promote the maintenance of adenoviral infectivity. In oneembodiment, adenoviral infectivity of the adenovirus polyol compositionis further defined as maintaining an infectivity of about 70% PFU/mL toabout 99.9% PFU/mL of the starting infectivity when stored for sixmonths at 4° centigrade. In preferred embodiments, adenoviralinfectivity is about 80% to 95% PFU/mL of the starting infectivity whenstored for six months at 4° centigrade.

[0023] In the context of the present invention, a polyol is defined as apolyhydric alcohol containing two or more hydroxyl groups. In certainembodiments, the polyol is glycerol, propylene glycol, polyethyleneglycol, sorbitol or mannitol, wherein the polyol concentration is fromabout 5% to about 30% (w/v). In other embodiments, the polyolconcentration is from about 10% to about 30%. In yet other embodiments,the polyol concentration is about 25%.

[0024] In a preferred embodiment, the aqueous pharmaceutical adenoviruscomposition comprises a polyol in an amount effective to promote themaintenance of adenoviral infectivity, wherein the polyol is glycerol,included in a concentration of from about 5% to about 30% (w/v).

[0025] In other embodiments, the aqueous pharmaceutical adenoviruscomposition comprising a polyol in an amount effective to promote themaintenance of adenoviral infectivity further comprises an excipient inaddition to the polyol, wherein the excipient is inositol, lactitol,xylitol, isomaltol, gelatin, agar, pectin, casein, dried skim milk,dried whole milk, silicate, carboxypolymethylene, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methhylcellulose,methylcellulose, sucrose, dextrose, lactose, trehalose, glucose,maltose, niacinamide, creatinine, monosodium glutamate dimethylsulfoxide, sweet whey solids, human serum albumin, bovine serum albumin,PEG, glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80.

[0026] In further defined embodiments, the aqueous pharmaceuticaladenovirus composition comprising a polyol further comprises in additionto the polyol at least a first and a second excipient, wherein thesecond excipient is different the first excipient, and the excipient isinositol, lactitol, xylitol, isomaltol, gelatin, agar, pectin, casein,dried skim milk, dried whole milk, silicate, carboxypolymethylene,hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethhylcellulose, methylcellulose, sucrose, dextrose, lactose,trehalose, glucose, maltose, niacinamide, creatinine, monosodiumglutamate dimethyl sulfoxide, sweet whey solids, human serum albumin,bovine serum albumin, PEG, glycine, arginine, proline, lysine, alanine,polyvinyl pyrrolidine, polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80.

[0027] In another embodiment of the present invention, a method for thepreparation of a long-term, storage stable adenovirus formulation,comprising the steps of providing adenovirus and combining theadenovirus with a solution comprising a buffer, a bulking agent, acryoprotectant and a lyoprotectant; and lyophilizing the solution,whereby lyophilization of the solution produces a freeze-dried cake ofthe adenovirus formulation that retains high infectivity and lowresidual moisture.

[0028] In particular embodiments, the bulking agent used for preparingthe freeze dried adenovirus formulation is mannitol, inositol, lactitol,xylitol, isomaltol, sorbitol, gelatin, agar, pectin, casein, dried skimmilk, dried whole milk, silcate, carboxypolymethylene, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methhylcellulose ormethylcellulose. In preferred embodiments, the bulking agent ismannitol, wherein mannitol comprises about 0.5% to about 8% (w/v) of theformulation.

[0029] In other embodiments, the cryoprotectant used for preparing thefreeze dried adenovirus formulation is sucrose, dextrose, lactose,trehalose, glucose, maltose, niacinamide, creatinine, monosodiumglutamate dimethyl sulfoxide or sweet whey solids. In preferredembodiments, the cryoprotectant is sucrose, wherein sucrose comprisesabout 2.5% to about 10% (w/v) of said formulation.

[0030] In further embodiments, the lyoprotectant used for preparing thefreeze dried adenovirus formulation is human serum albumin, bovine serumalbumin, PEG, glycine, arginine, proline, lysine, alanine, polyvinylpyrrolidine, polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80. In preferred embodiments, the lyoprotectant is human serumalbumin.

[0031] In other embodiments, the buffer used for preparing the freezedried adenovirus formulation is Tris-HCl, TES, HEPES, mono-Tris, brucinetetrahydrate, EPPS, tricine, or histidine, wherein the buffer is presentin the formulation at a concentration at about 1 mM to 50 mM. In onepreferred embodiment, the buffer used for preparing the freeze driedadenovirus formulation is Tris-HCl, wherein the Tris-HCl is included ina concentration of from about 1 mM to about 50 mM. In anotherembodiment, the Tris-HCl is included in a concentration of from about 5mM to about 20 mM. In still other embodiments, the freeze driedadenovirus formulation further comprises a salt selected from the groupconsisting of MgCl₂, MnCl₂, CaCl₂, ZnCl₂, NaCl and KCl.

[0032] In one embodiment, lyophilizing the adenovirus formulation iscarried out in the presence of an inert gas.

[0033] In certain embodiments, the method for preparing the freeze driedadenovirus formulation, wherein lyophilizing the solution comprises thesteps of, freezing the solution, subjecting the solution to a vacuum andsubjecting the solution to at least a first and a second drying cycle,whereby the second drying cycle reduces the residual moisture content ofthe freeze-dried cake to less than about 2%.

[0034] In another embodiment, a method for the preparation of along-term storage, stable adenovirus liquid formulation, comprising thesteps of providing adenovirus and combining the adenovirus with asolution comprising a buffer and a polyol, whereby the adenovirus liquidformulation retains high infectivity.

[0035] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various modifications withinthe spirit and scope of the invention will become apparent to thoseskilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0037]FIG. 1. Lyophilization Cycle of Adenovirus.

[0038]FIG. 2. Residual Moisture of Lyophilized Adenovirus AfterSecondary Drying at 10° C.

[0039]FIG. 3. Stability of Lyophilized Adenovirus after Secondary Dryingat 10° C.

[0040]FIG. 4. Residual Moisture of Lyophilized Adenovirus AfterSecondary Drying at 30° C.

[0041]FIG. 5. Stability of Lyophilized Adenovirus after Secondary Dryingat 30° C.

[0042]FIG. 6. HPLC Analysis of Lyophilized Adenovirus Stored at RoomTemperature.

[0043]FIG. 7. HPLC Analysis of Lyophilized Adenovirus Stored at 4° C.

[0044]FIG. 8. HPLC Analysis of Lyophilized Adenovirus Stored at −20° C.

[0045]FIG. 9A and FIG. 9B. Addition of DMSO to the formulation for anadenoviral vector increases the transduction efficiency. Human NSCLCxenografts were established on the flanks of nude mice. Animals receivedintratumoral injection of 2×10¹⁰ viral particles (vp) of Ad-βgalformulated in either PBS+glycerol (FIG. 9A and FIG. 9B, top panels) orin PBS+glycerol+5% DMSO (FIG. 9A and FIG. 9B, lower panels). Tumors wereexcised at either 24 (FIG. 9A) or 48 hours (FIG. 9B) post-injection andsectioned for histochemical analysis of reporter gene expression.Histochemical analysis was done on multiple sections from the tumorblock to analyze vector transduction and distribution. Two sections foreach formulation are illustrated: one from the tumor periphery (FIG. 9Aand FIG. 9B, left panels) and one from the center of the tumor (FIG. 9Aand FIG. 9B, right panels). In each section both transduction (asindicated by intensity of blue staining) and distribution (as indicatedby extent of blue staining) were improved by addition of DMSO to theformulation.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0046] The need for long-term stable virus formulations that can bestored at or above refrigerated temperatures without losing infectivityis highly desirable. Traditional methods of ultra-low temperaturestorage (≦60° C.) of virus preparations often limit the storage,transportation and clinical applications of viruses. The inventors havedeveloped optimal lyophilization formulations for freeze-dryingadenovirus in which the freeze-dried products maintain their stability(ie., infectivity of 60-100% of the starting infectivity) and have aresidual moisture of less than about 5% when stored for 6 months at 4°C.

[0047] In another embodiment, the inventors have developed long-termstable adenovirus formulations for storing adenovirus at 4° C. in aliquid form that maintains stability (i.e., infectivity of 60-100% ofthe starting infectivity) for at least 6 months.

[0048] A. Purification Techniques

[0049] A large scale process for the production and purification ofadenovirus is described in U.S. Ser. No. 08/975,519 filed Nov. 20, 1997(specifically incorporated herein by reference without disclaimer). Thisproduction process offers not only scalability and validatability butalso virus purity comparable to that achieved using CsCl gradientultracentrifugation. This process involves the preparation ofrecombinant adenovirus particles, the process comprising preparing aculture of producer cells by seeding producer cells into a culturemedium, infecting cells in the culture after mid-log phase growth with arecombinant adenovirus comprising a selected recombinant gene operablylinked to a promoter, harvesting recombinant adenovirus particles fromthe cell culture and removing contaminating nucleic acids. An importantaspect of this process is the removal of contaminating nucleic acidsusing nucleases. Exemplary nucleases include Benzonase®, Pulmozyme®; orany other DNase or RNase commonly used within the art.

[0050] Enzymes such as Benzonaze® degrade nucleic acid and have noproteolytic activity. The ability of Benzonase® to rapidly hydrolyzenucleic acids makes the enzyme ideal for reducing cell lysate viscosity.It is well known that nucleic acids may adhere to cell derived particlessuch as viruses. The adhesion may interfere with separation due toagglomeration, change in size of the particle or change in particlecharge, resulting in little if any product being recovered with a givenpurification scheme. Benzonase® is well suited for reducing the nucleicacid load during purification, thus eliminating the interference andimproving yield.

[0051] As with all endonuclease, Benzonase® hydrolyzes internalphosphodiester bonds between specific nucleotides. Upon completedigestion, all free nucleic acids present in solution are reduced tooligonucleotides 2 to 4 bases in length.

[0052] The present invention further employs a number of differentpurification techniques to purify viral vectors of the presentinvention. Such techniques include those based on sedimentation andchromatography and are described in more detail herein below.

[0053] 1. Density Gradient Centrifugation

[0054] There are two methods of density gradient centrifugation, therate zonal technique and the isopycnic (equal density) technique, andboth can be used when the quantitative separation of all the componentsof a mixture of particles is required. They are also used for thedetermination of buoyant densities and for the estimation ofsedimentation coefficients.

[0055] Particle separation by the rate zonal technique is based upondifferences in size or sedimentation rates. The technique involvescarefully layering a sample solution on top of a performed liquiddensity gradient, the highest density of which exceeds that of thedensest particles to be separated. The sample is then centrifuged untilthe desired degree of separation is effected, i.e., for sufficient timefor the particles to travel through the gradient to form discrete zonesor bands which are spaced according to the relative velocities of theparticles. Since the technique is time dependent, centrifugation must beterminated before any of the separated zones pellet at the bottom of thetube. The method has been used for the separation of enzymes, hormones,RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for theanalysis of size distribution of samples of polysomes and forlipoprotein fractionations.

[0056] The sample is layered on top of a continuous density gradientwhich spans the whole range of the particle densities which are to beseparated. The maximum density of the gradient, therefore, must alwaysexceed the density of the most dense particle. During centrifugation,sedimentation of the particles occurs until the buoyant density of theparticle and the density of the gradient are equal (i.e., wherep_(p)=p_(m) in equation 2.12). At this point no further sedimentationoccurs, irrespective of how long centrifugation continues, because theparticles are floating on a cushion of material that has a densitygreater than their own.

[0057] Isopycnic centrifugation, in contrast to the rate zonaltechnique, is an equilibrium method, the particles banding to form zoneseach at their own characteristic buoyant density. In cases where,perhaps, not all the components in a mixture of particles are required,a gradient range can be selected in which unwanted components of themixture will sediment to the bottom of the centrifuge tube whilst theparticles of interest sediment to their respective isopycnic positions.Such a technique involves a combination of both the rate zonal andisopycnic approaches.

[0058] Isopycnic centrifugation depends solely upon the buoyant densityof the particle and not its shape or size and is independent of time.Hence soluble proteins, which have a very similar density (e.g., p=1.3 gcm⁻³ in sucrose solution), cannot usually be separated by this method,whereas subcellular organelles (e.g., Golgi apparatus, p=1.11 g cm⁻³,mitochondria, p=1.19 g cm⁻³ and peroxisomes, p=1.23 g cm³¹ ³ in sucrosesolution) can be effectively separated.

[0059] As an alternative to layering the particle mixture to beseparated onto a preformed gradient, the sample is initially mixed withthe gradient medium to give a solution of uniform density, the gradient‘self-forming’, by sedimentation equilibrium, during centrifugation. Inthis method (referred to as the equilibrium isodensity method), use isgenerally made of the salts of heavy metals (e.g., cesium or rubidium),sucrose, colloidal silica or Metrizamide.

[0060] The sample (e.g., DNA) is mixed homogeneously with, for example,a concentrated solution of cesium chloride. Centrifugation of theconcentrated cesium chloride solution results in the sedimentation ofthe CsCl molecules to form a concentration gradient and hence a densitygradient. The sample molecules (DNA), which were initially uniformlydistributed throughout the tube now either rise or sediment until theyreach a region where the solution density is equal to their own buoyantdensity, i.e. their isopycnic position, where they will band to formzones. This technique suffers from the disadvantage that often very longcentrifugation times (e.g., 36 to 48 hours) are required to establishequilibrium. However, it is commonly used in analytical centrifugationto determine the buoyant density of a particle, the base composition ofdouble stranded DNA and to separate linear from circular forms of DNA.

[0061] Many of the separations can be improved by increasing the densitydifferences between the different forms of DNA by the incorporation ofheavy isotopes (e.g., ¹⁵N) during biosynthesis, a technique used byLeselson and Stahl to elucidate the mechanism of DNA replication inEsherichia coli, or by the binding of heavy metal ions or dyes such asethidium bromide. Isopycnic gradients have also been used to separateand purify viruses and analyze human plasma lipoproteins.

[0062] 2. Chromatography

[0063] Purification techniques are well known to those of skill in theart. These techniques tend to involve the fractionation of the cellularmilieu (e.g., density gradient centrifugation) to separate theadenovirus particles from other components of the mixture. Havingseparated adenoviral particles from the other components, the adenovirusmay be purified using chromatographic and electrophoretic techniques toachieve complete purification. Analytical methods particularly suited tothe preparation of a pure adenoviral particle of the present inventionare ion-exchange chromatography, size exclusion chromatography andpolyacrylamide gel electrophoresis. A particularly efficientpurification method to be employed in conjunction with the presentinvention is HPLC.

[0064] Certain aspects of the present invention concern thepurification, and in particular embodiments, the substantialpurification, of an adenoviral particle. The term “purified” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the adenoviral particle is purified to any degreerelative to its naturally-obtainable form. A purified adenoviralparticle therefore also refers to an adenoviral component, free from theenvironment in which it may naturally occur.

[0065] Generally, “purified” will refer to an adenoviral particle thathas been subjected to fractionation to remove various other components,and which composition substantially retains its expressed biologicalactivity. Where the term “substantially purified” is used, thisdesignation will refer to a composition in which the particle, proteinor peptide forms the major component of the composition, such asconstituting about 50% or more of the constituents in the composition.

[0066] Various methods for quantifying the degree of purification of aprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “—fold purification number”. The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

[0067] There is no general requirement that the adenovirus, always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater —fold purification than thesame technique utilizing a low pressure chromatography system. Methodsexhibiting a lower degree of relative purification may have advantagesin total recovery of protein product, or in maintaining the activity ofan expressed protein.

[0068] Of course, it is understood that the chromatographic techniquesand other purification techniques known to those of skill in the art mayalso be employed to purify proteins expressed by the adenoviral vectorsof the present invention. Ion exchange chromatography and highperformance liquid chromatography are exemplary purification techniquesemployed in the purification of adenoviral particles and are describedin further detail herein below.

[0069] a. Ion-Exchange Chromatography

[0070] The basic principle of ion-exchange chromatography is that theaffinity of a substance for the exchanger depends on both the electricalproperties of the material and the relative affinity of other chargedsubstances in the solvent. Hence, bound material can be eluted bychanging the pH, thus altering the charge of the material, or by addingcompeting materials, of which salts are but one example. Becausedifferent substances have different electrical properties, theconditions for release vary with each bound molecular species. Ingeneral, to get good separation, the methods of choice are eithercontinuous ionic strength gradient elution or stepwise elution. (Agradient of pH alone is not often used because it is difficult to set upa pH gradient without simultaneously increasing ionic strength.) For ananion exchanger, either pH and ionic strength are gradually increased orionic strength alone is increased. For a cation exchanger, both pH andionic strength are increased. The actual choice of the elution procedureis usually a result of trial and error and of considerations ofstability. For example, for unstable materials, it is best to maintainfairly constant pH.

[0071] An ion exchanger is a solid that has chemically bound chargedgroups to which ions are electrostatically bound; it can exchange theseions for ions in aqueous solution. Ion exchangers can be used in columnchromatography to separate molecules according to charge,; actuallyother features of the molecule are usually important so that thechromatographic behavior is sensitive to the charge density, chargedistribution, and the size of the molecule.

[0072] The principle of ion-exchange chromatography is that chargedmolecules adsorb to ion exchangers reversibly so that molecules can bebound or eluted by changing the ionic environment. Separation on ionexchangers is usually accomplished in two stages: first, the substancesto be separated are bound to the exchanger, using conditions that givestable and tight binding; then the column is eluted with buffers ofdifferent pH, ionic strength, or composition and the components of thebuffer compete with the bound material for the binding sites.

[0073] An ion exchanger is usually a three-dimensional network or matrixthat contains covalently linked charged groups. If a group is negativelycharged, it will exchange positive ions and is a cation exchanger. Atypical group used in cation exchangers is the sulfonic group, SO₃ ⁻. Ifan H⁺ is bound to the group, the exchanger is said to be in the acidform; it can, for example, exchange on H⁺ for one Na⁺ or two H⁺ for oneCa²⁺. The sulfonic acid group is called a strongly acidic cationexchanger. Other commonly used groups are phenolic hydroxyl andcarboxyl, both weakly acidic cation exchangers. If the charged group ispositive—for example, a quaternary amino group—it is a strongly basicanion exchanger. The most common weakly basic anion exchangers arearomatic or aliphatic amino groups.

[0074] The matrix can be made of various material. Commonly usedmaterials are dextran, cellulose, agarose and copolymers of styrene andvinylbenzene in which the divinylbenzene both cross-links thepolystyrene strands and contains the charged groups. Table 1 gives thecomposition of many ion exchangers.

[0075] The total capacity of an ion exchanger measures its ability totake up exchangeable groups per milligram of dry weight. This number issupplied by the manufacturer and is important because, if the capacityis exceeded, ions will pass through the column without binding. TABLE 1Matrix Exchanger Functional Group Tradename Dextran Strong CationicSulfopropyl SP-Sephadex Weak Cationic Carboxymethyl CM-Sephadex StrongAnionic Diethyl-(2- QAE-Sephadex hydroxypropyl)- aminoethyl Weak AnionicDiethylaminoethyl DEAE-Sephadex Cellulose Cationic CarboxymethylCM-Cellulose Cationic Phospho P-cel Anionic DiethylaminoethylDEAE-cellulose Anionic Polyethylenimine PEI-Cellulose AnionicBenzoylated- DEAE(BND)-cellulose naphthoylated, deiethylaminoethylAnionic p-Aminobenzyl PAB-cellulose Styrene- Strong Cationic Sulfonicacid AG 50 divinyl- benzene Strong Anionic AG 1 Strong Cationic Sulfonicacid + AG 501 + Tetramethylammonium Strong Anionic Acrylic Weak CationicCarboxylic Bio-Rex 70 Phenolic Strong Cationic Sulfonic acid Bio-Rex 40Expoxyamine Weak Anionic Tertiary amino AG-3

[0076] The available capacity is the capacity under particularexperimental conditions (i.e., pH, ionic strength). For example, theextent to which an ion exchanger is charged depends on the pH (theeffect of pH is smaller with strong ion exchangers). Another factor isionic strength because small ions near the charged groups compete withthe sample molecule for these groups. This competition is quiteeffective if the sample is a macromolecule because the higher diffusioncoefficient of the small ion means a greater number of encounters.Clearly, as buffer concentration increases, competition becomes keener.

[0077] The porosity of the matrix is an important feature because thecharged groups are both inside and outside the matrix and because thematrix also acts as a molecular sieve. Large molecules may be unable topenetrate the pores; so the capacity will decease with increasingmolecular dimensions. The porosity of the polystyrene-based resins isdetermined by the amount of cross-linking by the divinylbenzene(porosity decreases with increasing amounts of divinylbenzene). With theDowex and AG series, the percentage of divinylbenzene is indicated by anumber after an X - hence, Dowex 50-X8 is 8% divinylbenzene

[0078] Ion exchangers come in a variety of particle sizes, called meshsize. Finer mesh means an increased surface-to-volume ration andtherefore increased capacity and decreased time for exchange to occurfor a given volume of the exchanger. On the other hand, fine mesh meansa slow flow rate, which can increase diffusional spreading. The use ofvery fine particles, approximately 10 μm in diameter and high pressureto maintain an adequate flow is called high-performance or high-pressureliquid chromatography or simply HPLC.

[0079] Such a collection of exchangers having such differentproperties—charge, capacity, porosity, mesh—makes the selection of theappropriate one for accomplishing a particular separation difficult. Howto decide on the type of column material and the conditions for bindingand elution is described in the following Examples.

[0080] There are a number of choice to be made when employing ionexchange chromatography as a technique. The first choice to be made iswhether the exchanger is to be anionic or cationic. If the materials tobe bound to the column have a single charge (i.e., either plus orminus), the choice is clear. However, many substances (e.g., proteins,viruses), carry both negative and positive charges and the net chargedepends on the pH. In such cases, the primary factor is the stability ofthe substance at various pH values. Most proteins have a pH range ofstability (i.e., in which they do not denature) in which they are eitherpositively or negatively charged. Hence, if a protein is stable at pHvalues above the isoelectric point, an anion exchanger should be used;if stable at values below the isoelectric point, a cation exchanger isrequired.

[0081] The choice between strong and weak exchangers is also based onthe effect of pH on charge and stability. For example, if a weaklyionized substance that requires very low or high pH for ionization ischromatographed, a strong ion exchanger is called for because itfunctions over the entire pH range. However, if the substance is labile,weak ion exchangers are preferable because strong exchangers are oftencapable of distorting a molecule so much that the molecule denatures.The pH at which the substance is stable must, of course, be matched tothe narrow range of pH in which a particular weak exchanger is charged.Weak ion exchangers are also excellent for the separation of moleculeswith a high charge from those with a small charge, because the weaklycharged ions usually fail to bind. Weak exchangers also show greaterresolution of substances if charge differences are very small. If amacromolecule has a very strong charge, it may be impossible to elutefrom a strong exchanger and a weak exchanger again may be preferable. Ingeneral, weak exchangers are more useful than strong exchangers.

[0082] The Sephadex and Bio-gel exchangers offer a particular advantagefor macromolecules that are unstable in low ionic strength. Because thecross-links in these materials maintain the insolubility of the matrixeven if the matrix is highly polar, the density of ionizable groups canbe made several times greater than is possible with cellulose ionexchangers. The increased charge density means increased affinity sothat adsorption can be carried out at higher ionic strengths. On theother hand, these exchangers retain some of their molecular sievingproperties so that sometimes molecular weight differences annul thedistribution caused by the charge differences; the molecular sievingeffect may also enhance the separation.

[0083] Small molecules are best separated on matrices with small poresize (high degree of cross-linking) because the available capacity islarge, whereas macromolecules need large pore size. However, except forthe Sephadex type, most ion exchangers do not afford the opportunity formatching the porosity with the molecular weight.

[0084] The cellulose ion exchangers have proved to be the best forpurifying large molecules such as proteins and polynucleotides. This isbecause the matrix is fibrous, and hence all functional groups are onthe surface and available to even the largest molecules. In may caseshowever, beaded forms such as DEAE-Sephacel and DEAE-Biogel P are moreuseful because there is a better flow rate and the molecular sievingeffect aids in separation.

[0085] Selecting a mesh size is always difficult. Small mesh sizeimproves resolution but decreases flow rate, which increases zonespreading and decreases resolution. Hence, the appropriate mesh size isusually determined empirically.

[0086] Because buffers themselves consist of ions, they can alsoexchange, and the pH equilibrium can be affected. To avoid theseproblems, the rule of buffers is adopted: use cationic buffers withanion exchangers and anionic buffers with cation exchangers. Becauseionic strength is a factor in binding, a buffer should be chosen thathas a high buffering capacity so that its ionic strength need not be toohigh. Furthermore, for best resolution, it has been generally found thatthe ionic conditions used to apply the sample to the column (theso-called starting conditions) should be near those used for eluting thecolumn.

[0087] b. High Performance Liquid Chromatography

[0088] High performance liquid chromatography(HPLC) is characterized bya very rapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

[0089] B. Viral Formulation

[0090] Retrovirus, adenovirus, adeno-associated virus, and herpessimplex virus are the most commonly used viruses in gene therapy(Robbins and Ghivizzani; 1998). It is contemplated in the presentinvention that the preparation of long-term stable adenovirus vectorsthat can be stored at or above refrigerated temperatures would be usefulas gene therapy vectors. Viral particles must maintain their structuralintegrity to remain infective and biologically active for use as genetherapy vectors. Current virus formulations do not readily make itfeasible to store or transport viral vector at or above refrigeratedtemperatures without significant loss of viral infectivity.

[0091] The present invention describes long-term stable adenovirusformulations that can be stored at 4° C. for periods up to 6 months. Inone embodiment of the present invention, adenovirus preparations areformulated for lyophilization and long-term storage at 4° C. asfreeze-dried adenovirus. In another embodiment, the adenovirus isprepared as a liquid formulation that is long-term stable at 4° C. Animportant aspect of both the lyophilized and liquid adenovirusformulations is the addition of at least one or more compounds thatimprove the long-term, storage stability of the adenovirus.

[0092] The term “compound” in the context of the present inventionincludes pharmaceutically acceptable carriers such as bulking agents,cryoprotectants, lyoprotectants, preservatives, solvents, solutes andany additional pharmaceutical agents well known in the art. Bufferingagents and other types of pH control can also be added simultaneously inorder to provide for maximum buffering capacity for the adenovirusformulation. For example, pH changes that deviate from physiologicalconditions often result in irreversible aggregation of proteins (Wetzel,1992) and viral capsids (Misselwitz et al., 1995) due to complete orpartial denaturation of the protein. Thus, buffering agents areparticularly important for virus preparations that aggregate or denatureat sub-optimal pH ranges.

[0093] 1. Lyophilized Formulations

[0094] The formulation of lyophilized, long-term storage stableadenovirus in the present invention requires the presence of one or moreexcipients. More particularly, for optimal long-term stability oflyophilized adenovirus formulations, a bulking agent and one or moreprotectants are desirable. It is well known in the art that loss invirus infectivity often is directly related to denaturation, selfassociation and aggregation of the viral particles (Misselwitz et al.,1995; Vanlandschoot et al., 1998; Sagrera et al., 1998; Lu et al.,1998). In fact, the E. coli heat shock proteins GroEL/GroES have beenshown to both stabilize viral particles from denaturation andaggregation during high stress cellular conditions and to facilitatecapsid assembly during non-stressed, normal cellular conditions (Polissiet al., 1995; Nakonechny and Teschke, 1998).

[0095] The use of bulking agents, cryoprotectants, lyoprotectants andsalts in the present invention are included in the formulation oflyophilized adenovirus to improve long-term stability (i.e. infectivity)of the adenovirus freeze-dried products. The stabilizing effect of thecryoprotectant sucrose against irreversible denaturation and aggregationhas been described previously as an excluded volume effect (Hall et al.,1995). Similarly, bulking agents, cryo- and lyoprotectants such aspolyacrylamide gels, agaorse gels, dextran and polyethylene glycol (PEG)have demonstrated enhanced stabilities of proteins and nucleic acids inpart by excluded volume effects (Fried and Bromberg, 1997; Vossen andFried, 1997). The exact mechanistic details of excluded volume effectsare still not clear. A currently accepted theory is that many of thesecompounds result in the preferential hydration of protein molecules(i.e. volume of exclusion), which tends to stabilize the native versusthe denatured conformation of proteins, and therefore preventsaggregation. In addition, the presence of low concentrations ofcosolvents (e.g., salts) result in charge screening of proteins andviral protein coats increasing their solubility in water.

[0096] The use of bulking agents, cryoprotectants, lyoprotectants andsalts in the present invention are contemplated and demonstratedexperimentally to improve the storage stability of lyophilizedadenovirus products. In one embodiment, a bulking agent and protectantsare combined with a buffer comprising adenovirus.

[0097] Bulking agents, cryoprotectants and lyoprotectants are well knownin the art (Lueckel et al., 1998; Herman et al., 1994; Croyle et al.,1998; Corveleyn and Remon, 1996). Bulking agents considered in thepresent invention are mannitol, inositol, lactitol, xylitol, isomaltol,sorbitol, gelatin, agar, pectin, casein, dried skim milk, dried wholemilk, silcate, carboxypolymethylene, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methhylcellulose, methylcelluloseand other bulking agents well known in the art. Cryoprotectantsconsidered are sucrose, dextrose, lactose, trehalose, glucose, maltose,niacinamide, creatinine, monosodium glutamate, dimethyl sulfoxide, sweetwhey solids, as well as other known cryoprotectants. Lyoprotectantscontemplated for use in the present invention are human serum albumin,bovine serum albumin, PEG, glycine, arginine, proline, lysine, alanine,polyvinyl pyrrolidine, polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20and Tween-80. Certain lyoprotectants are also classified ascryoprotectants and vice versa. For the purpose of the presentinvention, cryoprotectants and lyoprotectants are represented asindependent classes of compounds. However, this classification is onlyfor clarity of the invention and should not limit the person skilled inthe art from using any excipient that stabilizes the adenovirusformulation. In other embodiments of the present invention, the termexcipient encompasses bulking agents, cryo- and lyoprotectants. Incertain embodiments, salts are included in the formulation in additionto the aforementioned excipients. The following salts are considered foruse in the present invention MgCl₂, MnCl₂, CaCl₂, ZnCl₂, NaCl, and KCl,but should not preclude the use of other salts that improve stability ofthe adenovirus formulation.

[0098] In other embodiments of the invention, the lyophilized adenovirusformulation is dried in the presence of an inert gas or a combination ofinert gasses. The purging of the lyophilization vessel with an inert gasor gasses, the presence of the inert gas or gasses during lyophilizationof the adenovirus solution and during the capping of the lyophilizationvial after the drying step, are contemplated to minimize the deleteriouseffects of O₂. It is known that residual O₂ leads to oxidation anddegradation of proteins. It is contemplated that purging and capping ofthe freeze-dried adenovirus product improves the long-term storagestability of the adenovirus product. The use of antioxidants such asβ-mercapto ethanol, DTT, citric acid and the like may also be consideredfor use in formulations.

[0099] An important aspect of the lyophilization process is a seconddrying cycle. The second drying cycle is at a temperature of 30° C. forat least 3.5 hours, which is demonstrated to reduce the residualmoisture of the adenovirus freeze-dried product to less than 2% waterimmediately after drying. It is contemplated that the reduced residualmoisture improves the long-term storage stability of the adenovirusfreeze-dried product. Longer drying times up to 20 hours are thuscontemplated to further reduce residual moisture.

[0100] 2. Liquid Formulations

[0101] The formulation of liquid, long-term storage stable adenovirus inthe present invention requires the presence of a polyol. A polyol is apolyhydric alcohol containing two or more hydroxyl groups. For optimallong-term stability of liquid adenovirus formulations in the presentinvention, glycerol is used. In particular embodiments of the invention,the presence 20% glycerol results in adenovirus stability (80% PFU/mL)for periods of time at least up to 6 months days when stored at 4° C.

[0102] Glycerol (glycerin) is one of the oldest and most widely usedexcipients in pharmaceutical products. It is a clear, colorless liquidwhich is miscible with water and alcohol. Glycerol is hygroscopic,stable to mild acidic and basic environments and can be sterilized attemperatures up to 150° C. It is well known as both a taste masking andcryoprotective agent, as well as an antimicrobial agent. It has goodsolubilizing power and is a commonly used solvent in parenteralformulations. It is considered to be one of the safest excipients usedsince it is metabolized to glucose, or to substances which are involvedwith triglyceride synthesis or glycolysis (Frank et al., 1981). It is aGRAS listed excipient and typically used at levels up to 50% inparenteral formulations.

[0103] The stabilizing effects of glycerol on protein structure is wellknown in the art (Hase et al., 1998; Juranville et al., 1998). Severalstudies indicate that glycerol has a similar effect of viral particles.For example, when competent Haemophilus influenza bacteria were exposedto purified phage and plated for transfectants, a 100-fold increase intransfectants was observed when 32% glycerol was present in the solution(Stuy, 1986) In yet another study, glycerol was demonstrated to preservethe integrity of vaccinia virus (Slonin and Roslerova, 1969).

[0104] Other polyols contemplated for use in the present invention arepolyethylene glycol, propylene glycol, sorbitol, mannitol, and the like.Polyethylene glycols are polymers of ethylene oxide with the generalformula:

HO—CH₂—(CH₂—O—CH₂)_(n)—CH₂OH

[0105] where n represents the number of oxyethylene groups. The PEG'sare designated by a numerical value, which is indicative of the averagemolecular weight for a given grade. Molecular weights below 600 areliquids, and molecular weights above 1000 are solids at roomtemperature. These polymers are readily soluble in water, which makethem quite useful for parenteral dosage forms. Only PEG 400 and PEG 300are utilized in parenteral products, typically at concentrations up to30% v/v. These polymers are generally regarded as non-toxic andnon-irritating. There are numerous reviews regarding the pharmaceuticaland toxicological characteristics of these polyols (Smyth et al., 1950;Rowe and Wolf, 1982, Swarbrick and Boylan, 1990).

[0106] Propylene glycol, a dihydroxy alcohol, is one of the more commonsolvents encountered in pharmaceutical cosolvent formulations, for bothparenteral and non-parenteral dosage forms. PG is generally regarded asnon-toxic. It is more hygroscopic than glycerin, and has excellentsolubilizing power for a wide variety of compounds. In addition, it hasexcellent bacteriocidal and preservative properties (Heine et al, 1950).PG is metabolized to carbon dioxide and water via lactic and pyruvicacid intermediates and, therefore, not prone to the severe toxicities.

[0107] Sorbitol and mannitol are hexahydric alcohols, consisting ofwhite, crystalline powders, that are soluble in water. Both are commonlyused excipients in pharmaceutical products with little or no toxicityassociated, as approved by the FDA for food use. The mechanistics ofsorbitol and mannitol protein and viral stabilization is still notcompletely understood. Current theories suggest at least part of theeffect is osmotic diuretic (Vanholder, et al., 1984; de Rizzo, et al.,1988). The use of the polyols described above are considered exemplary,but should not limit the skilled artist from selecting other polyolsthat confer viral stability for liquid formulations.

[0108] It is also contemplated, in addition to a polyol in the liquidformulation, that one or possibly two excipients may also be included.Excipients considered for use in the present invention are inositol,lactitol, xylitol, isomaltol, gelatin, agar, pectin, casein, dried skimmilk, dried whole milk, silcate, carboxypolymethylene, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methhylcellulose,methylcellulose, sucrose, dextrose, lactose, trehalose, glucose,maltose, niacinamide, creatinine, monosodium glutamate, dimethylsulfoxide, sweet whey solids, human serum albumin, bovine serum albumin,glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20and Tween-80. The choice of a particular excipient is dependent in someinstances on the desired properties of the viral formulation.

[0109] In particular embodiments, dimethyl sulfoxide (DMSO) iscontemplated for use in the present invention. DMSO has beendemonstrated to enhance- the infectivity of adenovirus preparations byincreasing the efficiency of gene transfer (Chikada and Jones, 1999).For example, the infectivity of adenovirus type 2 DNA in 293 cells wasincreased up to five-fold by the brief treatment of cell monolayers with25% DMSO (Chinnadurai et al., 1978) The stabilization of virus particlesvia DMSO also has been reported (Wallis and Melnick, 1968). The presentinventors demonstrate that the intratumoral administration of Ad-p53 isimproved when DMSO is added to 5 or 10% (see FIG. 9). Adenovirus studiesvia intravesical administration indicate that an adenoviral vector maybe stable in up to 50% DMSO (WO 98/35554). In other embodiments, apolyol contemplated for use in the present invention as an enhancer ofadenovirus gene transduction is a polyoxyalkene (U.S. Pat. No.5,552,309, specifically incorporate herein by reference in itsentirety).

[0110] Thus in particular embodiments, an adenoviral formulationaccording to the present invention may also contain DMSO. Theconcentration for intratumoral administration may contain from about 2%to 67% DMSO, preferably from about 5% to 20%. The concentration forintravesical administration may contain from about 2% to 67% DMSO,preferably from about 20% to 50%. The concentration for topicaladministration may contain from about 2% to 67% DMSO, preferably fromabout 10% to 40%. The concentration for intra-articular administrationmay contain from about 2% to 67% DMSO, preferably, from about 5% to 40%.The concentration for systemic administration may contain from about 2%to 75% DMSO, preferably from about 50% to 67%.

[0111] Adenovirus polyol formulations of the invention may futurecomprise a polyoxamer, such as Polyoxamer 407, at concentrations of fromabout 0.5% to 20%, preferably from about 10% to 20%. The formulationstorage stable adenovirus may also contain from about 5% to 40%dimethylacetamide, preferably from about 10% to 25%, Or it may containfrom about 10% to 50% of a polyethylene glycol, such as polyethyleneglycol 400, preferably from about 15% to 50%. Of course, the formulationof said adenovirus also may contain combinations of the abovecomponents.

[0112] C. Viral Transformation

[0113] The present invention employs, in one example, adenoviralinfection of cells in order to generate therapeutically significantvectors. Typically, the virus will simply be exposed to the appropriatehost cell under physiologic conditions, permitting uptake of the virus.Though adenovirus is exemplified, the present methods may beadvantageously employed with other viral vectors, as discussed below.

[0114] 1. Viral Infection

[0115] a. Adenovirus

[0116] One method for delivery of the recombinant DNA involves the useof an adenovirus expression vector. Although adenovirus vectors areknown to have a low capacity for integration into genomic DNA, thisfeature is counterbalanced by the high efficiency of gene transferafforded by these vectors. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to ultimately express arecombinant gene construct that has been cloned therein.

[0117] The vector comprises a genetically engineered form of adenovirus.Knowledge of the genetic organization or adenovirus, a 36 kb, linear,double-stranded DNA virus, allows substitution of large pieces ofadenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz,1992). In contrast to retrovirus, the adenoviral infection of host cellsdoes not result in chromosomal integration because adenoviral DNA canreplicate in an episomal manner without potential genotoxicity. Also,adenoviruses are structurally stable, and no genome rearrangement hasbeen detected after extensive amplification.

[0118] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target-cell range and high infectivity. Both ends of theviral genome contain 100-200 base pair inverted repeats (ITRs), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP, (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

[0119] In a current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

[0120] Generation and propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses E1 proteins (Graham etal., 1977). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, 1978), the current adenovirus vectors, with thehelp of 293 cells, carry foreign DNA in either the E1, the D3 or bothregions (Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone.

[0121] Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

[0122] Racher et al. (1995) have disclosed improved methods forculturing 293 cells and propagating adenovirus. In one format, naturalcell aggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

[0123] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

[0124] As stated above, the typical vector according to the presentinvention is replication defective and will not have an adenovirus E1region. Thus, it will be most convenient to introduce the transformingconstruct at the position from which the E1-coding sequences have beenremoved. However, the position of insertion of the construct within theadenovirus sequences is not critical to the invention. Thepolynucleotide encoding the gene of interest may also be inserted inlieu of the deleted E3 region in E3 replacement vectors as described byKarlsson et al. (1986) or in the E4 region where a helper cell line orhelper virus complements the E4 defect.

[0125] Adenovirus growth and manipulation is known to those of skill inthe art, and exhibits broad host range in vitro and in vivo. This groupof viruses can be obtained in high titers, e.g., 10⁹-10¹¹ plaque-formingunits per ml, and they are highly infective. The life cycle ofadenovirus does not require integration into the host cell genome. Theforeign genes delivered by adenovirus vectors are episomal and,therefore, have low genotoxicity to host cells. No side effects havebeen reported in studies of vaccination with wild-type adenovirus (Couchet al., 1963; Top et al., 1971), demonstrating their safety andtherapeutic potential as in vivo gene transfer vectors.

[0126] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Adenoviralvectors also have been described for treatment of certain types ofcancers (U.S. Pat. No. 5,789,244, specifically incorporated herein byreference in its entirety). Animal studies have suggested thatrecombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld etal., 1992), muscle injection (Ragot etal.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

[0127] b. Retrovirus

[0128] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

[0129] In order to construct a retroviral vector, a nucleic acidencoding a gene of interest is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann etal., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

[0130] Concern with the use of defective retrovirus vectors is thepotential appearance of wild-type replication-competent virus in thepackaging cells. This can result from recombination events in which theintact sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

[0131] c. Adeno-Associated Virus

[0132] Adeno-associated virus (AAV) is an attractive vector system foruse in the present invention as it has a high frequency of integrationand it can infect nondividing cells, thus making it useful for deliveryof genes into mammalian cells in tissue culture (Muzyczka, 1992). AAVhas a broad host range for infectivity (Tratschin, et al., 1984;Laughlin, et al., 1986; Lebkowski, et al., 1988; McLaughlin, et al.,1988), which means it is applicable for use with the present invention.Details concerning the generation and use of rAAV vectors are describedin U.S. Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, eachincorporated herein by reference.

[0133] Studies demonstrating the use of AAV in gene delivery includeLaFace et al. (1988); Zhou et al. (1993); Flotte et al. (1993); andWalsh et al. (1994). Recombinant AAV vectors have been used successfullyfor in vitro and in vivo transduction of marker genes (Kaplitt et al.,1994; Lebkowski et al., 1988; Samulski et al., 1989; Shelling and Smith,1994; Yoder et al., 1994; Zhou et al., 1994; Hermonat and Muzyczka,1984; Tratschin et al., 1985; McLaughlin et al., 1988) and genesinvolved in human diseases (Flotte et al., 1992; Luo et al., 1994; Ohiet al., 1990; Walsh et al., 1994; Wei et al., 1994). Recently, an AAVvector has been approved for phase I human trials for the treatment ofcystic fibrosis.

[0134] AAV is a dependent parvovirus in that it requires coinfectionwith another virus (either adenovirus or a member of the herpes virusfamily) to undergo a productive infection in cultured cells (Muzyczka,1992). In the absence of coinfection with helper virus, the wild-typeAAV genome integrates through its ends into human chromosome 19 where itresides in a latent state as a provirus (Kotin et al., 1990; Samulski etal., 1991). rAAV, however, is not restricted to chromosome 19 forintegration unless the AAV Rep protein is also expressed (Shelling andSmith, 1994). When a cell carrying an AAV provirus is superinfected witha helper virus, the AAV genome is “rescued” from the chromosome or froma recombinant plasmid, and a normal productive infection is established(Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990;Muzyczka, 1992).

[0135] Typically, recombinant AAV (rAAV) virus is made by cotransfectinga plasmid containing the gene of interest flanked by the two AAVterminal repeats (McLaughlin et al., 1988; Samulski et al., 1989; eachincorporated herein by reference) and an expression plasmid containingthe wild-type AAV coding sequences without the terminal repeats, forexample pIM45 (McCarty etal., 1991; incorporated herein by reference).The cells are also infected or transfected with adenovirus or plasmidscarrying the adenovirus genes required for AAV helper function. rAAVvirus stocks made in such fashion are contaminated with adenovirus whichmust be physically separated from the rAAV particles (for example, bycesium chloride density centrifugation). Alternatively, adenovirusvectors containing the AAV coding regions or cell lines containing theAAV coding regions and some or all of the adenovirus helper genes couldbe used (Yang et al., 1994a; Clark et al., 1995). Cell lines carryingthe rAAV DNA as an integrated provirus can also be used (Flotte et al.,1995).

[0136] d. Other Viral Vectors

[0137] Other viral vectors may be employed as constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) andherpesviruses may be employed. They offer several attractive featuresfor various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwaland Sugden, 1986; Couparet al., 1988; Horwich et al., 1990).

[0138] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. Chang et al. recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and pre-surface codingsequences. It was cotransfected with wild-type virus into an avianhepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

[0139] Also contemplated for use in the present invention is a fairlynew class of viruses termed oncolytic virus (Pennisi, 1998). Some of theviruses included in this group are reovirus, the genetically modifiedadenovirus OYNX-015 and CN706. These oncolytic viruses, which have notbeen genetically altered to prevent their replication, destroy certaintypes of cancer cells by multiplying and spreading, killing only thecancer cells. Each of the above oncolytic viruses are proposed tooperate via different pathways involved in cancers.

[0140] For example, human reovirus requires an activated Ras signalingpathway for infection of cultured cells. Thus, in certain tumors with anoveractive ras gene, reovirus readily replicates. In a study onreovirus, severe combined immune deficient mice bearing tumorsestablished from v-erbB-transformed murine NIH 3T3 cells or human U87glioblastoma cells were treated with the virus. A single intratumoralinjection of virus resulted in regression of tumors in 65% to 80% of themice. Treatment of immune-competent C3H mice bearing tumors establishedfrom a ras-transformed C3H-10T1/2 cells also resulted in tumorregression, although a series of injections were required (Coffey etal., 1998)

[0141] 2. Vectors and Regulatory Signals

[0142] Vectors of the present invention are designed, primarily, totransform cells with a 10 gene under the control of regulated eukaryoticpromoters (i.e., inducible, repressable, tissue specific). Also, thevectors usually will contain a selectable marker if, for no otherreason, to facilitate their production in vitro. However, selectablemarkers may play an important role in producing recombinant cells andthus a discussion of promoters is useful here. Table 2 and Table 3below, list inducible promoter elements and enhancer elements,respectively. TABLE 2 Inducible Elements Element Inducer References MTII Phorbol Ester (TFA) Palmiter et al., 1982; Haslinger and Heavy metalsKarin, 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al,1987; Karin ® , 1987; Angel et al., 1987b; MeNeall et al., 1989 MMTV(mouse Glucocorticoids Huang etal., 1981; Lee et al., 1981; mammarytumor virus) Majors and Varmus, 1983; Chandler et al., 1983; Lee et al.,1984; Fonta et al., 1985; Sakai et al., 1986 β-Interferon poly(rI)XTavemier et al., 1983 poly(rc) Adenovirus 5 E2 Ela Imperiale and Nevins,1984 Collagenase Phorbol Ester (TPA) Angle et al., 1987a StromelysinPhorbol Ester (TPA) Angle et al., 1987b SV40 Phorbol Ester (TFA) Angelet al., 1987b Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23 187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al.,1989 Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2KbInterferon Blanar et al., 1989 HSP70 Ela, SV40 Large T Taylor et al,1989; Taylor and Antigen Kingston, 1990a,b Proliferin Phorbol Ester-TPAMordacq and Linzer, 1989 Tumor Necrosis Factor EMA Hensel et al., 1989Thyroid Stimulating Thyroid Hormone Chatterjee et al., 1989 Hormone αGene

[0143] TABLE 3 Other Promoter/Enhancer Elements Promoter/EnhancerReferences immunoglobulin Heavy Chain Hanerji et al., 1983; Gilles etal., 1983; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986,1987; Imler et al., 1987; Weinberger et al., 1988; Kiledjian et al.,1988; Porton et al., 1990 Immunoglobulin Light Chain Queen andBaltimore, 1983; Picard and Schaffner, 1984 T-Cell Receptor Luria etal., 1987, Winoto and Baltimore, 1989; Redondo et al., 1990 HLA DQ α andDQ β Sullivan and Peterlin, 1987 β-Interferon Goodbourn et al., 1986;Fujita et al., 1987; Goodboum and Maniatis, 1985 Interleukin-2 Greene etal., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRα Sherman et al.,1989 β-Actin Kawamoto et al., 1988; Ng et al., 1989 Muscle CreatineKinase Jaynes et al., 1988; Horlick and Benfield, 1989; Johnson et al.,1989a Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Omitz etal., 1987 Metallothionen Karin et al., 1987; Culotta and Hamer, 1989Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin GenePinkert et al., 1987, Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere and Tilghman, 1989 t-Globin Bodine and Ley, 1987;Perez-Stable and Constantini, 1990 β-Globin Trudel and Constantini, 1987e-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsch et al.,1990 (NCAM) a_(1-Antitrypain) Latimer et at., 1990 H2B (TH2B) HistoneHwang et al., 1990 Mouse or Type I Collagen Ripe et al., 1989Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) RatGrowth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrookeet at., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-DerivedGrowth Factor Pech et al., 1989 Duchenne Muscular Dystrophy Klamut etal., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh andLockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbraand Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek etal., 1987; Kuhl et al., 1987 Schaffneret al., 1988 PolyomaSwartzendruber and Lehman, 1975; Vasseur et al., 1980; Katinka et al.,1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers etal., 1984; Hen et al., 1986; Satake et al., 1988; Campbell andVillarreal, 1988 Retroviruses Kriegler and Botchan, 1982, 1983; Levinsonet al., 1982; Kriegler et al., 1983, 1984a,b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman and Rotter, 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos andWilkie, 1983; Spalholz et al., 1985; Lusky and Botchan, 1986; Cripe etal., 1987; Gloss et al., 1987; Hirochika et al., 1987, Stephens andHentschel, 1987; Glue et al., 1988 Hepatitis B Virus Bulla and Siddiqui,1986; Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau andLee, 1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber andCullan, 1988; Jakobovits et al., 1988; Feng and Holland, 1988; Takebe etal., 1988; Rowen et al., 1988; Berkhout et al., 1989; Laspia et al.,1989; Sharp and Marciniak, 1989; Braddock et al., 1989 CytomegalovirusWeber et al., 1984; Boshart et al., 1985; Foecking and Hofstetter, 1986Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989

[0144] Another signal that may prove useful is a polyadenylation signal(hGH, BGH, SV40).

[0145] The use of internal ribosome binding sites (IRES) elements areused to create multigene, or polycistronic, messages. IRES elements areable to bypass the ribosome scanning model of 5′-methylatedcap-dependent translation and begin translation at internal sites(Pelletier and Sonenberg, 1988). IRES elements from two members of thepicomavirus family (polio and encephalomyocarditis) have been described(Pelletier and Sonenberg, 1988), as well an IRES from a mammalianmessage (Macejak and Sarnow, 1991). IRES elements can be linked toheterologous open reading frames. Multiple open reading frames can betranscribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame isaccessible to ribosomes for efficient translation. Multiple genes can beefficiently expressed using a single promoter/enhancer to transcribe asingle message.

[0146] As discussed above, in certain embodiments of the invention, acell may be identified and selected in vitro or in vivo by including amarker in the expression construct. Such markers confer an identifiablechange to the cell permitting easy identification of cells containingthe expression construct. Usually, the inclusion of a drug selectionmarker aids in cloning and in the selection of transformants, forexample, genes that confer resistance to neomycin, puromycin,hygromycin, DHFR, GPT, zeocin, tetracycline and histidinol are usefulselectable markers. Alternatively, enzymes such as herpes simplex virusthymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may beemployed.

[0147] The promoters and enhancers that control the transcription ofprotein encoding genes in eukaryotic cells are composed of multiplegenetic elements. The cellular machinery is able to gather and integratethe regulatory information conveyed by each element, allowing differentgenes to evolve distinct, often complex patterns of transcriptionalregulation.

[0148] The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator proteins.

[0149] At least one module in each promoter functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV 40 late genes, a discrete element overlying thestart site itself helps to fix the place of initiation.

[0150] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between elements is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenelements can be increased to 50 bp apart before activity begins todecline. Depending on the promoter, it appears that individual elementscan function either co-operatively or independently to activatetranscription.

[0151] Enhancers were originally detected as genetic elements thatincreased transcription from a promoter located at a distant position onthe same molecule of DNA. This ability to act over a large distance hadlittle precedent in classic studies of prokaryotic transcriptionalregulation. Subsequent work showed that regions of DNA with enhanceractivity are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

[0152] The basic distinction between enhancers and promoters isoperational. An enhancer region as a whole must be able to stimulatetranscription at a distance; this need not be true of a promoter regionor its component elements. On the other hand, a promoter must have oneor more elements that direct initiation of RNA synthesis at a particularsite and in a particular orientation, whereas enhancers lack thesespecificities. Aside from this operational distinction, enhancers andpromoters are very similar entities.

[0153] Promoters and enhancers have the same general function ofactivating transcription in the cell. They are often overlapping andcontiguous, often seeming to have a very similar modular organization.Taken together, these considerations suggest that enhancers andpromoters are homologous entities and that the transcriptional activatorproteins bound to these sequences may interact with the cellulartranscriptional machinery in fundamentally the same way.

[0154] In any event, it will be understood that promoters are DNAelements which when positioned functionally upstream of a gene leads tothe expression of that gene. Most transgene constructs of the presentinvention are functionally positioned downstream of a promoter element.

[0155] D. Engineering of Viral Vectors

[0156] In certain embodiments, the present invention further involvesthe manipulation of viral vectors. Such methods involve the use of avector construct containing, for example, a heterologous DNA encoding agene of interest and a means for its expression, replicating the vectorin an appropriate helper cell, obtaining viral particles producedtherefrom, and infecting cells with the recombinant virus particles. Thegene could simply encode a protein for which large quantities of theprotein are desired, i.e., large scale in vitro production methods.Alternatively, the gene could be a therapeutic gene, for example totreat cancer cells, to express immunomodulatory genes to fight viralinfections, or to replace a gene's function as a result of a geneticdefect. In the context of the gene therapy vector, the gene will be aheterologous DNA, meant to include DNA derived from a source other thanthe viral genome which provides the backbone of the vector. Finally, thevirus may act as a live viral vaccine and express an antigen of interestfor the production of antibodies they are against. The gene may bederived from a prokaryotic or eukaryotic source such as a bacterium, avirus, a yeast, a parasite, a plant, or even an animal. The heterologousDNA also may be derived from more than one source, i.e., a multigeneconstruct or a fusion protein. The heterologous DNA may also include aregulatory sequence which may be derived from one source and the genefrom a different source.

[0157] 1. Therapeutic Genes

[0158] p53 currently is recognized as a tumor suppressor gene(Montenarh, 1992). High levels of mutant p53 have been found in manycells transformed by chemical carcinogenesis, ultraviolet radiation, andseveral viruses, including SV40. The p53 gene is a frequent target ofmutational inactivation in a wide variety of human tumors and is alreadydocumented to be the most frequently-mutated gene in common humancancers (Mercer, 1992). It is mutated in over 50% of human NSCLC(Hollestein et al., 1991) and in a wide spectrum of other tumors.

[0159] The p53 gene encodes a 393-amino-acid phosphoprotein that canform complexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are generally minute by comparison with transformed cells or tumortissue. Interestingly, wild-type p53 appears to be important inregulating cell growth and division. Overexpression of wild-type p53 hasbeen shown in some cases to be anti-proliferative in human tumor celllines. Thus, p53 can act as a negative regulator of cell growth(Weinberg, 1991) and may directly suppress uncontrolled cell growth ordirectly or indirectly activate genes that suppress this growth. Thus,absence or inactivation of wild-type p53 may contribute totransformation. However, some studies indicate that the presence ofmutant p53 may be necessary for full expression of the transformingpotential of the gene.

[0160] Wild-type p53 is recognized as an important growth regulator inmany cell types. Missense mutations are common for the p53 gene and areknown to occur in at least 30 distinct codons, often creating dominantalleles that produce shifts in cell phenotype without a reduction tohomozygosity. Additionally, many of these dominant negative allelesappear to be tolerated in the organism and passed on in the germ line.Various mutant alleles appear to range from minimally dysfunctional tostrongly penetrant, dominant negative alleles (Weinberg, 1991).

[0161] Casey and colleagues have reported that transfection of DNAencoding wild-type p53 into two human breast cancer cell lines restoresgrowth suppression control in such cells (Casey et a., 1991). A similareffect has also been demonstrated on transfection of wild-type, but notmutant, p53 into human lung cancer cell lines (Takahasi et al., 1992).p53 appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 is not detrimental to normal cellswith endogenous wild-type p53. Thus, such constructs might be taken upby normal cells without adverse effects. It is thus proposed that thetreatment of p53-associated cancers with wild-type p53 expressionconstructs will reduce the number of malignant cells or their growthrate. Furthermore, recent studies suggest that some p53 wild-type tumorsare also sensitive to the effects of exogenous p53 expression.

[0162] The major transitions of the eukaryotic cell cycle are triggeredby cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase4 (CDK4), regulates progression through the G₁ phase. The activity ofthis enzyme may be to phosphorylate Rb at late G₁. The activity of CDK4is controlled by an activating subunit, D-type cyclin, and by aninhibitory subunit, e.g. p16^(INK)4, which has been biochemicallycharacterized as a protein that specifically binds to and inhibits CDK4,and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serranoet al, 1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano,1993), deletion of this gene may increase the activity of CDK4,resulting in hyperphosphorylation of the Rb protein. p16 also is knownto regulate the function of CDK6.

[0163] p16^(INK4) belongs to a newly described class of CDK-inhibitoryproteins that also includes p16^(B), p21^(WAF1, CIP1, SDI1) andp27^(KIP1). The p16^(INK4) gene maps to 9p21, a chromosome regionfrequently deleted in many tumor types. Homozygous deletions andmutations of the p16^(INK4) gene are frequent in human tumor cell lines.This evidence suggests that the p16^(INK4) gene is a tumor suppressorgene. This interpretation has been challenged, however, by theobservation that the frequency of the p16^(INK4) gene alterations ismuch lower in primary uncultured tumors than in cultured cell lines(Caldas et al., 1994; Cheng et al., 1994; Hussussian et al., 1994; Kambet al., 1994a; Kamb et al., 1994b; Mori et al., 1994; Okamoto et al,1994; Nobori et al., 1995; Orlow et al, 1994; Arap et al., 1995).Restoration of wild-type p16^(INK4) function by transfection with aplasmid expression vector reduced colony formation by some human cancercell lines (Okamoto, 1994; Arap, 1995).

[0164] C-CAM is expressed in virtually all epithelial cells (Odin andObrink, 1987). C-CAM, with an apparent molecular weight of 105 kD, wasoriginally isolated from the plasma membrane of the rat hepatocyte byits reaction with specific antibodies that neutralize cell aggregation(Obrink, 1991). Recent studies indicate that, structurally, C-CAMbelongs to the immunoglobulin (Ig) superfamily and its sequence ishighly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti,1989). Using a baculovirus expression system, Cheung et al. (1993a;1993b and 1993c) demonstrated that the first Ig domain of C-CAM iscritical for cell adhesion activity.

[0165] Cell adhesion molecules, or CAMs are known to be involved in acomplex network of molecular interactions that regulate organdevelopment and cell differentiation (Edelman, 1985). Recent dataindicate that aberrant expression of CAMs may be involved in thetumorigenesis of several neoplasms; for example, decreased expression ofE-cadherin, which is predominantly expressed in epithelial cells, isassociated with the progression of several kinds of neoplasms (Edelmanand Crossin, 1991; Frixen et al., 1991; Bussemakers et al., 1992;Matsura et al., 1992; Umbas et al., 1992). Also, Giancotti and Ruoslahti(1990) demonstrated that increasing expression of α₅β₁, integrin by genetransfer can reduce tumorigenicity of Chinese hamster ovary cells invivo. C-CAM now has been shown to suppress tumor growth in vitro and invivo.

[0166] Other tumor suppressors that may be employed according to thepresent invention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,zac1, p73, BRCA1, VHL, FCC, MMAC1, MCC, p16, p21, p57, C-CAM, p27 andBRCA2. Inducers of apoptosis, such as Bax, Bak, Bc1-X_(s), Bik, Bid,Harakiri, Ad E1B, Bad and ICE-CED3 proteases, similarly could find useaccording to the present invention.

[0167] Various enzyme genes are of interest according to the presentinvention. Such enzymes include cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinaseand human thymidine kinase.

[0168] Hormones are another group of gene that may be used in thevectors described herein. Included are growth hormone, prolactin,placental lactogen, luteinizing hormone, follicle-stimulating hormone,chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin (ACTH), angiotensin I and II, β-endorphin,β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I,galanin, gastric inhibitory peptide (GIP), glucagon, insulin,lipotropins, neurophysins, somatostatin, calcitonin, calcitonin generelated peptide (CGRP), β-calcitonin gene related peptide, hypercalcemiaof malignancy factor (1-40), parathyroid hormone-related protein(107-139) (PTH-rP), parathyroid hormone-related protein (107-111)(PTH-rP), glucagon-like peptide (GLP-1), pancreastatin, pancreaticpeptide, peptide YY, PHM, secretin, vasoactive intestinal peptide (VIP),oxytocin, vasopressin (AVP), vasotocin, enkephalinamide, metorphinamide,alpha melanocyte stimulating hormone (alpha-MSH), atrial natriureticfactor (5-28) (ANF), amylin, amyloid P component (SAP-1), corticotropinreleasing hormone (CRH), growth hormone releasing factor (GHRH),luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, substanceK (neurokinin A), substance P and thyrotropin releasing hormone (TRH).

[0169] Other classes of genes that are contemplated to be inserted intothe vectors of the present invention include interleukins and cytokines.Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11 IL-12, GM-CSF and G-CSF.

[0170] Examples of diseases for which the present viral vector would beuseful include, but are not limited to, adenosine deaminase deficiency,human blood clotting factor IX deficiency in hemophilia B, and cysticfibrosis, which would involve the replacement of the cystic fibrosistransmembrane receptor gene. The vectors embodied in the presentinvention could also be used for treatment of hyperproliferativedisorders such as rheumatoid arthritis or restenosis by transfer ofgenes encoding angiogenesis inhibitors or cell cycle inhibitors.Transfer of prodrug activators such as the HSV-TK gene can be also beused in the treatment of hyperploiferative disorders, including cancer.

[0171] 2. Antisense constructs Oncogenes such as ras, myc, neu, raf,erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl also are suitabletargets. However, for therapeutic benefit, these oncogenes would beexpressed as an antisense nucleic acid, so as to inhibit the expressionof the oncogene. The term “antisense nucleic acid” is intended to referto the oligonucleotides complementary to the base sequences ofoncogene-encoding DNA and RNA. Antisense oligonucleotides, whenintroduced into a target cell, specifically bind to their target nucleicacid and interfere with transcription, RNA processing, transport and/ortranslation. Targeting double-stranded (ds) DNA with oligonucleotideleads to triple-helix formation; targeting RNA will lead to double-helixformation.

[0172] Antisense constructs may be designed to bind to the promoter andother control regions, exons, introns or even exon-intron boundaries ofa gene. Antisense RNA constructs, or DNA encoding such antisense RNAs,may be employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject. Nucleic acid sequences comprising“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform only combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA.

[0173] As used herein, the terms “complementary” or “antisensesequences” mean nucleic acid sequences that are substantiallycomplementary over their entire length and have very few basemismatches. For example, nucleic acid sequences of fifteen bases inlength may be termed complementary when they have a complementarynucleotide at thirteen or fourteen positions with only single or doublemismatches. Naturally, nucleic acid sequences which are “completelycomplementary” will be nucleic acid sequences which are entirelycomplementary throughout their entire length and have no basemismatches.

[0174] While all or part of the gene sequence may be employed in thecontext of antisense construction, statistically, any sequence 17 baseslong should occur only once in the human genome and, therefore, sufficeto specify a unique target sequence. Although shorter oligomers areeasier to make and increase in vivo accessibility, numerous otherfactors are involved in determining the specificity of hybridization.Both binding affinity and sequence specificity of an oligonucleotide toits complementary target increases with increasing length. It iscontemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more base pairs will be used. One can readilydetermine whether a given antisense nucleic acid is effective attargeting of the corresponding host cell gene simply by testing theconstructs in vitro to determine whether the endogenous gene's functionis affected or whether the expression of related genes havingcomplementary sequences is affected.

[0175] In certain embodiments, one may wish to employ antisenseconstructs which include other elements, for example, those whichinclude C-5 propyne pyrimidines. Oligonucleotides which contain C-5propyne analogues of uridine and cytidine have been shown to bind RNAwith high affinity and to be potent antisense inhibitors of geneexpression (Wagner et al., 1993).

[0176] As an alternative to targeted antisense delivery, targetedribozymes may be used. The term “ribozyme” refers to an RNA-based enzymecapable of targeting and cleaving particular base sequences in oncogeneDNA and RNA. Ribozymes can either be targeted directly to cells, in theform of RNA oligo-nucleotides incorporating ribozyme sequences, orintroduced into the cell as an expression construct encoding the desiredribozymal RNA. Ribozymes may be used and applied in much the same way asdescribed for antisense nucleic acids.

[0177] 3. Antigens for Vaccines

[0178] Other therapeutics genes might include genes encoding antigenssuch as viral antigens, bacterial antigens, fungal antigens or parasiticantigens. Viruses include picomavirus, coronavirus, togavirus,flavirviru, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,arenvirus, reovirus, retrovirus, papovavirus, parvovirus, herpesvirus,poxvirus, hepadnavirus, and spongiform virus. Preferred viral targetsinclude influenza, herpes simplex virus 1 and 2, measles, small pox,polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms,helminths, . Also, tumor markers, such as fetal antigen or prostatespecific antigen, may be targeted in this manner. Preferred examplesinclude HIV env proteins and hepatitis B surface antigen. Administrationof a vector according to the present invention for vaccination purposeswould require that the vector-associated antigens be sufficientlynon-immunogenic to enable long term expression of the transgene, forwhich a strong immune response would be desired. Preferably, vaccinationof an individual would only be required infrequently, such as yearly orbiennially, and provide long term immunologic protection against theinfectious agent.

[0179] 4. Control Regions

[0180] In order for the viral vector to effect expression of atranscript encoding a therapeutic gene, the polynucleotide encoding thetherapeutic gene will be under the transcriptional control of a promoterand a polyadenylation signal. A “promoter” refers to a DNA sequencerecognized by the synthetic machinery of the host cell, or introducedsynthetic machinery, that is required to initiate the specifictranscription of a gene. A polyadenylation signal refers to a DNAsequence recognized by the synthetic machinery of the host cell, orintroduced synthetic machinery, that is required to direct the additionof a series of nucleotides on the end of the mRNA transcript for properprocessing and trafficking of the transcript out of the nucleus into thecytoplasm for translation. The phrase “under transcriptional control”means that the promoter is in the correct location in relation to thepolynucleotide to control RNA polymerase initiation and expression ofthe polynucleotide.

[0181] The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work-, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

[0182] At least one module in each promoter functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

[0183] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the tk promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either cooperatively or independentlyto activate transcription.

[0184] E. Pharmaceutical Compositions

[0185] In certain embodiments, the present invention also concernsformulations of a viral composition for administration to a mammal. Itwill also be understood that, if desired, the viral compositionsdisclosed herein may be administered in combination with other agents aswell, such as, e.g., various pharmaceutically-active agents. As long asthe compositions do not cause a significant adverse effect upon contactwith the target cells or host tissues, there is virtually no limit toother components which may also be included.

[0186] The formulation of pharmaceutically-acceptable excipients andcarrier solutions are well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

[0187] 1. Injectable Compositions and Delivery

[0188] The pharmaceutical compositions disclosed herein may beadministered parenterally, intravenously, intramuscularly, or evenintraperitoneally as described in U.S. Pat. No. 5,543,158; U.S. Pat. No.5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporatedherein by reference in its entirety). Solutions of the active compoundsas free base or pharmacologically acceptable salts may be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

[0189] Typically, these formulations may contain at least about 0.1% ofthe active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared in such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

[0190] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial adantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0191] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

[0192] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0193] The compositions disclosed herein may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like.

[0194] As used herein, “carrier” includes any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0195] The phrase “pharmaceutically-acceptable” refers to molecularentities and compositions that do not produce an allergic or similaruntoward reaction when administered to a human. The preparation of anaqueous composition that contains a protein as an active ingredient iswell understood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

[0196] 2. Oral Compositions and Delivery

[0197] Alternatively, the pharmaceutical compositions disclosed hereinmay be delivered via oral administration to an animal, and as such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

[0198] The active compounds may even be incorporated with excipients andused in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitzet al., 1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

[0199] For oral administration the compositions of the present inventionmay alternatively be incorporated with one or more excipients in theform of a mouthwash, dentifrice, buccal tablet, oral spray, orsublingual orally-administered formulation. For example, a mouthwash maybe prepared incorporating the active ingredient in the required amountin an appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as those containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, including: gels,pastes, powders and slurries, or added in a therapeutically effectiveamount to a paste dentifrice that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants, or alternativelyfashioned into a tablet or solution form that may be placed under thetongue or otherwise dissolved in the mouth.

[0200] 3. Nasal Delivery

[0201] The administration of agonist pharmaceutical compositions byintranasal sprays, inhalation, and/or other aerosol delivery vehicles isalso considered. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No.5,804,212 (each specifically incorporated herein by reference in itsentirety), and delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

EXAMPLES

[0202] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Materials and Methods

[0203] Lyophilizer A Dura-stop μp lyophilizer (FTSsystems) with inprocess sample retrieving device was used. The lyophilizer is equippedwith both thermocouple vacuum gauge and capacitance manometer for vacuummeasurement. Condenser temperature is programmed to reach to −80° C.Vials were stoppered at the end of each run with a build-in mechanicalstoppering device.

[0204] Residual Moisture Measurement

[0205] Residual moisture in freeze dried product was analyzed by aKarl-Fisher type coulometer (Mettler DL37, KF coulometer).

[0206] HPLC Analysis

[0207] HPLC analysis of samples was done on a Beckman Gold HPLC system.

[0208] Vials and Stoppers Borosilicate 3 ml with 13 mm opening lyo vialsand their corresponding butyl rubber stoppers (both from Wheaton) wereused for both lyophilization and liquid formulation development. Thestoppered vials were capped with Flip-off aluminum caps using a cappingdevice (LW3 12 Westcapper, The West Company).

Example 2 Lyophilization: Initial Cycle and Formulation Development

[0209] There are three main process variables that can be programmed toachieve optimal freeze-drying. Those are shelf temperature, chamberpressure, and lyophilization step duration time. To avoid cake collapse,shelf temperature need to be set at temperatures 2-3° C. below the glasstransition or eutectic temperature of the frozen formulation. Both theglass transition and eutectic temperatures of a formulation can bedetermined by differential scanning calorimetry (DSC) analysis. Chamberpressure is generally set at below the ice vapor pressure of the frozenformulation. The ice vapor pressure is dependent on the shelftemperature and chamber pressure. Too high a chamber pressure willreduce the drying rate by reducing the pressure differential between theice and the surrounding, while too low a pressure will also slow downdrying rate by reducing the heat transfer rate from the shelf to thevials. The development of a lyophilization cycle is closely related withthe formulation and the vials chosen for lyophilization. The goal atthis stage was to develop a somewhat conservative cycle to be able tosuccessfully freeze dry a number of different formulations. Thedeveloped cycles and formulations will be further optimized when virusesare formulated in the formulations. Formulation excipient selection wasbased on the classical excipients found in most lyophilizedpharmaceuticals. The excipients in a lyophilization formulation shouldprovide the functions of bulking, cryoprotection, and lyoprotection. Theexcipients chosen were mannitol (bulking agent), sucrose (cryo- andlyoprotectant), and human serum albumin (HSA, lyoprotectant). Theseexcipients were formulated in 10 mM Tris+1 mM MgCl₂, pH=7.50 at variouspercentages and filled into the 3 ml vials at a fill volume of 1 ml. Tostart with, a preliminary cycle was programmed to screen a variety offormulations based on the criteria of residual moisture and physicalappearance after drying. The cycle used is plotted in FIG. 1. Extensivescreening was carried out by variation of the percentages of theindividual excipients. Table 4 shows briefly some of the results. TABLE4 Evaluation of Different Formulations Under the Same Cycle FormulationM %/S %/HSA % Appearance Moisture (% weight) 10/5/0.5  good cake  0.895/5/0.5 good cake 1.5 3/5/0.5 loose cake (partial collapse) 3.4 1/5/0.5no cake (collapse) 6.4

[0210] The results suggest that a minimum amount of 3% mannitol isrequired in the formulation in order to achieve pharmaceutically elegantcake. The percentages of sucrose in the formulation were also examined.No significant effect on freeze-drying was observed at sucroseconcentrations of ≦10%. HSA concentration was kept constant to 0.5%during the initial screening stage.

[0211] After the evaluation of the formulations, freeze-drying cycle wasoptimized by changing the shelf temperature, chamber vacuum and theduration of each cycle step. Based on the extensive cycle optimization,the following cycle (cycle #14) was used for further viruslyophilization development.

[0212] 1. Load sample at room temperature onto shelf.

[0213] 2. Set shelf temperature to −45° C. and freeze sample. Step time2 h.

[0214] 3. Set shelf temperature at −45° C., turn vacuum pump and setvacuum at 400 mT. Step time 5 h.

[0215] 4. Set shelf temperature at −35° C., set vacuum at 200 mT. Steptime 13 h.

[0216] 5. Set shelf temperature at −22° C., set vacuum at 100 mT. Steptime 15 h.

[0217] 6. Set shelf temperature at −10° C., set vacuum at 100 mT. Steptime 5 h.

[0218] 7. Set shelf temperature at 10° C., set vacuum at 100 mT. Steptime 4 h.

[0219] 8. Vial stoppering under vacuum.

Example 3 Cycle and Formulation Development With Virus in Formulation

[0220] Effect of Sucrose Concentration in Formulation. Cycle andformulation were further optimized according to virus recovery afterlyophilization analyzed by both HPLC and plaque forming unit (PFU)assays. Table 5 shows the virus recoveries immediate after drying indifferent formulations using the above drying cycle. Variation of thepercentage of sucrose in the formulation had significant effect on virusrecoveries. TABLE 5 Recoveries of Virus After Lyophilization FormulationM %/S %/HSA % Appearance Residual moisture Recovery (%) 6/0/0.5 goodcake  0.44%  0  6/3.5/0.5 good cake 2.2% 56 6/5/0.5 good cake 2.5% 816/6/0.5 good cake 2.7% 120  6/7/0.5 good cake 2.8% 120  6/8/0.5 goodcake 3.3% 93 6/9/0.5 good cake 3.7% 120 

[0221] Residual moisture in the freeze-dried product increased as thesucrose percentage increased. A minimum sucrose concentration of 5% isrequired in the formulation to maintain a good virus recovery afterlyophilization. Similar sucrose effects in formulation that had 5%instead of 6% mannitol were observed. However, good virus recoveryimmediately after drying does not necessary support a good long-termstorage stability. As a result, formulations having 4 different sucroseconcentrations of 6, 7, 8, and 9%, were incorporated for furtherevaluation.

[0222] Effect of HSA in Formulation. The contribution of HSAconcentrations in the formulation on virus recovery after drying wasexamined using the same freeze drying cycle. Table 6 shows the results.TABLE 6 Effects of HSA Concentration on Lyophilization Formulation M %/S%/HSA % Appearance Residual moisture Recovery (%) 6/7/0 Good cake 0.98 83   6/7/0.5 Good cake 1.24 120 6/7/2 Good cake 1.5  110 6/7/5 Goodcake 1.7  102

[0223] The results indicate that inclusion of HSA in the formulation hadpositive effect on virus recovery after drying. Concentrations higherthan 0.5% did not further improve the virus recovery post drying. As aresult, 0.5% HSA is formulated in all the lyophilization formulations.

[0224] Cycle Optimization. As indicated in Table 5, relatively highresidual moistures were present in the dried product. Although there hasnot been a known optimal residual moisture for freeze dried viruses, itcould be beneficial for long term storage stability to further reducethe residual moisture in the dried product. After reviewing of thedrying cycle, it was decided to increase the secondary dryingtemperature from 10° C. to 30° C. without increasing the total cycletime. As indicated in Table 7, significant reduction in residualmoisture had been achieved in all the formulations without negativeeffects on virus recoveries. With the improved drying cycle, residualmoisture was less than 2% in all the formulations immediately afterdrying. It is expected that the reduced residual moisture will improvethe long-term storage stability of the dried product. TABLE 7 Effects ofSecondary Drying Temperature on Lyophilization Secondary drying at 10°C. Secondary drying Residual at 30° C. Formulation moisture RecoveryResidual M %/S %/HSA % (w %) (%) moisture Recovery 6/6/0.5 2.2 100  0.893 6/7/0.5 2.5 86 1.1 100  6/8/0.5 2.7 83 1.3 87 6/9/0.5 3.3 93 1.5 965/6/0.5 2.3 110  1.0 94 5/7/0.5 2.7 88 1.2 85 5/8/0.5 3.5 97 1.6 885/9/0.5 4   90 1.9 86

[0225] N₂ Backfilling (Blanketing). Lyophilization was done similarly asabove except that dry N₂ was used for gas bleeding for pressure controlduring the drying and backfilling at the end of the cycle. At the end ofa drying run, the chamber was filled with dry N₂ to about 80%atmospheric pressure. Subsequently, the vials were stoppered. Nodifference was noticed between the air and N₂ blanketing runs immediateafter drying. However, if oxygen present in the vial during airbackfilling causes damaging effect (oxidation) on the virus orexcipients used during long-term storage, backfilling with dry N₂ islikely to ameliorate the damaging effects and improve long term storagestability of the virus.

[0226] Removal of Glycerol From Formulation. During the preparation ofvirus containing formulations, stock virus solution was added to thepre-formulated formulations at a dilution factor of 10. Because of thepresence of 10% glycerol in the stock virus solution, 1% glycerol wasintroduced into the formulations. To examine any possible effect of thepresence of 1% glycerol on lyophilization, a freeze drying run wasconducted using virus diafiltered into the formulation of 5%(M)/7%(S)/0.5% (HSA). Diafiltration was done with 5 vol. of bufferexchange using a constant volume buffer exchange mode to ensure adequateremoval of residual glycerol (99% removal). After diafiltration, virussolution was filled into vials and then lyophilized similarly. Table 8shows the lyophilization results. TABLE 8 Lyophilization withoutGlycerol Formulation M %/S %/HSA % Residual moisture Recovery (%)5/7/0.5 1.0 80

[0227] No significant difference after freeze drying was observedbetween formulations with and without 1% glycerol. Possible implicationsof this change on long term storage will be evaluated.

Example 4 Long Term Storage Stability Study

[0228] Adp53 virus lyophilized under different formulations anddifferent cycles was placed at −20° C., 4° C., and room temperature (RT)under dark for long term storage stability evaluation. Parametersmeasured during the stability study were PFU, HPLC viral particles,residual moisture, and vacuum inside vial. The plan is to be able toevaluate virus stability at various conditions for up to one-yearstorage. Table 6 shows the data after 12-month storage with secondarydrying at 10° C. without N₂ blanketing. Lyophilized virus is stable atboth −20° C. and 4° C. storage for up to 12 months. However, virus wasnot stable at room temperature storage. More than 50% loss ininfectivity was observed at RT after 1-month storage. The reason for thequick loss of infectivity at RT is not clear. However, it is likely thatRT is above the glass transition temperature of the dried formulationand resulting in the accelerated virus degradation. A differentialscanning caloremitry (DSC) analysis of the formulation could providevery useful information. Pressure change inside the vials during storagewas not detected, which indicates that the vials maintained theirintegrity. The slight increase in residual moisture during storage canbe attributed to the release of moisture from the rubber stopper intothe dried product. TABLE 9 Secondary Drying at 10° C. Formulation Set 10(6-9) PFU × 10⁹/ml HPLC Viral Particle (× 10¹⁰/ml) Water Content (W %)Date* Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 4/11/97 5.5 6.0 5.8 6.5 24.524.6 24.9 26.7 2.2 2.5 2.7 3.3 5/15/97a 7.6 7.1 7.5 8.1 22.4 25.6 26.828.5 2.2 2.5 2.8 3.3 5/15/97^(b) 6.6 6.3 6.5 10.0 22.0 23.0 24.0 27.52.4 2.6 3.0 3.4 5/15/96^(c) 7.1 7.1 6.7 3.3 14.5 16.5 6.2 4.2 2.7 2.93.2 3.5 7/18/97^(a) 6.8 6.4 6.8 7.2 28.7 28.9 28.6 31.2 2.3 2.5 2.8 3.37/18/97^(b) 6.0 5.8 7.3 9.0 25.0 26.6 27.6 31.1 2.5 2.8 3.0 3.67/18/97^(c) 1.2 0.8 4.0 1.4 0.9 1.8 0.7 0.7 2.7 2.9 3.0 3.4 10/22/97^(a)7.9 7.5 7.9 7.8 25.5 25.0 25.4 26.2 2.4 2.6 2.8 3.1 10/22/97^(b) 6.8 6.85.8 8.0 22.0 23.0 24.7 24.2 2.7 2.9 3.2 3.6 10/22/97^(c) <0.01 <0.01<0.01 <0.01 N.D. N.D. N.D. N.D. 2.7 2.9 3.1 3.4 4/16/98^(a) 6.0 5.8 7.17.2 19.3 20.3 23.5 26.1 2.4 2.6 3.0 3.4 4/16/98^(b) 5.4 7.2 6.1 6.3 21.722.8 22.9 24.6 2.9 3.1 3.3 3.8 4/16/98^(c) 0.0003 0.001 0.0007 0.001N.D. N.D. N.D. N.D. 2.7 2.9 3.1 3.4 Controls 4/11/97 5.5 7.0 7.0 7.035.5 35.8 36.0 36.9 Formulation Set 11 (6-9) PFU × 10⁹/ml HPLC ViralParticle (× 10¹⁰/ml) Water Content (W %) Date* Set 11-6 Set 11-7 Set11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set11-8 Set 11-9 5/2/97 7.0 6.0 6.3 5.8 28.5 28.8 28.4 29.5 2.3 2.7 3.5 4.06/20/97^(a) 6.2 6.6 6.9 65 26.4 25.0 27.0 27.3 2.2 2.8 34 4.66/20/97^(b) 6.1 6.0 6.5 6.5 24.1 22.1 25.6 26.6 2.5 2.8 3.5 4.86/20/97^(c) 3.3 3.0 1.0 <0.1 20.5 17.4 5.2 9.1 2.7 3.1 3.5 4.78/18/97^(a) 8.0 7.2 7.5 7.6 21.6 21.8 25.3 24.9 2.3 2.8 3.7 4.98/18/97^(b) 8.0 7.3 8.0 8.0 22.7 22.7 24.9 25.0 2.6 3 3.9 4.28/18/97^(c) <0.1 <0.1 <0.1 <0.1 N.D. N.D. 0.2 13.1 2.7 3.0 3.5 4.410/22/97^(a) 79 7.5 7.9 6.7 21.0 22.0 25.1 24.0 2.4 3.0 3.9 4.410/22/97^(b) 6.0 6.9 6.8 7.3 21.4 22.0 23.1 23.1 2.6 3.0 3.3 4.610/22/97^(c) <0.01 <0.01 <0.01 <0.015 N.D. N.D. N.D. 9.0 2.7 2.9 3.9 5.05/8/98^(a) 8.3 7.5 8.0 8.7 19.0 18.2 19.9 21.1 2.6 3.1 4.0 4.65/8/98^(b) 7.0 7.1 7.8 6.5 17.3 17.1 18.2 17.8 2.8 3.2 4.1 5.15/8/98^(c) 0.00033 0.000065 0.00045 0.000016 N.D. N.D. N.D. N.D. 2.7 2.94.0 4.9 Controls 5/2/97 6.4 6.8 6.5 6.5 37.7 35.7 37.3 36.0

[0229] TABLE 10 Secondary Drying at 30° C. Without N₂ BlanketingFormulation Set 10 (6-9) PFU × 10⁹/ml HPLC Viral Particle (× 10¹⁰/ml)Water Content (W %) Date* Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 5/15/976.5 5.6 6.1 6.0 18.0 18.6 21.9 23.3 0.8 1.1 1.3 1.5 6/20/97^(b) 5.4 5.65.5 5.5 14.6 14.9 17.2 16.6 0.8 1.2 1.5 1.6 6/20/97^(c) 4.5 5.0 5.5 6.010.8 11.8 15.0 15.4 1.3 1.4 1.6 1.9 8/18/97^(b) 7.0 6.7 6.8 7.0 15.317.1 17.9 17.7 1.3 1.5 1.5 1.7 8/18/97^(c) 2.4 2.2 4.8 5.8 4.3 7.2 11.714.2 1.3 1.6 1.7 2.1 11/20/97^(b) 5.5 5.5 5.3 5.7 21.9 21.9 27.2 26.4l.1 1.4 1.6 1.9 11/20/97^(c) 0.5 0.9 2.3 3.1 1.5 6.3 8.8 13.5 1.3 1.71.8 2.2 5/14/98^(ab) 4.9 4.7 5.4 6.5 9.7 11.9 12.6 14.2 1.2 1.6 2.2 1.45/14/98^(c) 0.000006 0.00006 0.00004 0.000024 N.D. N.D. N.D. N.D. 1.41.6 1.3 2.0 Controls 5/15/97 7.0 5.6 7.0 7.0 31.2 30.6 31.6 31.4Formulation Set 11 (6-9) PFU × 10⁹/ml HPLC Viral Particle (× 10¹⁰/ml)Water Content (W %) Date* Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 5/22/977.5 6.3 7.3 6.5 17.4 16.6 20.3 24.7 1.0 1.2 1.6 1.9 6/20/97^(b) 5.5 6.36.0 7.5 14.8 16.1 17.5 21.1 1.2 1.3 1.7 1.8 6/20/97^(c) 5.0 6.0 6.0 7.512.6 14.9 17.2 20.3 1.4 1.6 1.9 2.0 8/18/97^(b) 6.3 6.7 68 7.5 15.7 17.218.5 22.6 1.2 1.5 1.8 1.9 8/18/97^(c) 3.3 4.5 5.5 7.0 7.4 10.5 15.8 21.21.6 1.7 1.9 2.2 11/20/97^(b) 5.3 5.6 5.3 6.6 22.6 26.4 30.0 35.0 1.2 1.41.9 1.9 11/20/97^(c) 0.8 1.9 3.0 0.1 3.2 9.6 18.3 1.3 1.6 1.7 2.0 2.15/14/98^(ab) 6.7 7.2 6.9 7.6 12.4 13.9 15.5 18.5 1.3 1.6 2.0 2.25/14/98^(c) 0.0013 0.00005 0.00031 0.00045 N.D. N.D. N.D. N.D. 1.6 1.81.6 2.0 Controls 5/22/97 8.0 7.4 8.3 7.6 26.7 27.6 27.5 32.4

[0230] TABLE 11 Secondary Drying at 30° C. With N₂ BlanketingFormulation Set 10 (6-9) + Adp53 PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) Water Content (W %) Date* Set 10-6 Set 10-7 Set 10-8 Set 10-9Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-96/13/97 3.4 4.3 4.1 4.2 16.0 16.5 16.1 18.1 0.8 1.1 1.3 1.4 7/18/97^(b)6.3 6.3 6.0 6.0 17.9 19.5 19.9 20.6 0.9 1.2 1.4 1.6 7/18/97^(c) 4.1 5.55.0 5.5 11.4 15.5 18.2 20.6 1.2 1.4 1.7 1.8 9/16/97^(b) 4.2 5.5 4.5 5.115.3 16.1 16.4 17.8 1.0 1.3 1.5 1.7 9/16/97^(c) 0.7 1.2 5.0 4.0 2.9 5.09.5 13.0 1.3 1.5 1.8 2.0 12/4/97^(b) 5.5 5.3 5.4 5.9 16.1 16.2 18.1 18.51.1 1.4 1.6 1.7 12/4/97^(c) 0.3 0.5 2.5 3.4 N.D. 1.7 4.7 8.8 1.4 1.6 1.82.0 6/29/98^(ab) 3.8 5.1 5.3 5.4 10.6 10.8 12.0 12.9 1.3 1.5 1.8 1.96/29/98^(c) 0.00003 0.00006 0.0001 0.0001 N.D. N.D. N.D. N.D. 1.4 1.61.7 1.8 Controls 6/13/97 4.7 3.8 5.5 6.2 26.0 26.2 27.4 27.5 ContinuedFormulation set 11 (6-9) + Adp53 PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) Water Content (W %) Date* Set 11-6 Set 11-7 Set 11-8 Set 11-9Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-96/13/97 3.4 4.2 3.6 4.4 16.1 16.3 18.4 19.3 0.9 1.3 1.8 1.9 7/18/97^(b)5.5 6.2 6.5 6.2 18.0 19.5 23.0 23.9 1.0 1.4 1.8 2.1 7/18/97^(c) 3.7 6.06.7 7.3 13.7 18.7 21.8 22.8 1.3 1.7 2.0 2.2 9/16/97^(b) 3.9 4 4.6 6 15.617.3 19.5 20.6 1.3 1.5 1.9 2.1 9/16/97^(c) 0.78 2.2 4.0 5.3 3.6 6.8 13.814.6 1.5 1.9 2.3 2.4 12/4/97^(b) 4.6 5.3 8.0 6.1 15.7 18.2 21.4 21.6 1.21.6 2.1 2.2 12/4/97^(c) 0.4 0.6 0.3 0.01 N.D. N.D. 1.7 N.D. 1.6 1.8 2.12.1 6/29/98^(ab) 4.9 5.0 5.4 6.4 11.4 14.2 13.7 16.0 1.5 1.7 2.1 2.66/29/98^(c) 0.0001 0.00015 0.00085 0.0012 N.D. N.D. N.D. N.D. 1.6 1.72.2 2.3 Controls 6/13/97 4.5 5.0 4.0 5.0 26.5 26.9 26.6 27.1

[0231] TABLE 12 Aqueous Formulation Set #1 Date PFU × 10⁹/ml (StorageConds.) 10%-G 5%-S + 5%-HSA 5%-S + 1%-PEG 5%-T + 1%-PEG 8/1/97 5.8 4.74.3 4.4 8/28/97 (4° C., N₂) 5.8 5.8 6.4 6.3 8/28/97 (4° C., Air) 5.0 5.96.0 5.9 8/28/97 (R.T., N₂) 4.4 4.8 5.0 6.0 8/28/97 (R.T., Air) 4.3 5.05.0 5.6 10/30/97 (4° C., N₂) 3.8 4.0 4.7 3.8 10/30/97 (4° C., Air) 3.04.1 3.7 4.7 10/30/97 (R.T., N2) 1.5 3.4 3.5 3.6 10/30/97 (R.T., Air) 1.53.6 2.2 3.1 1/12/98 (4° C., N₂) 3.2 4.1 3.3 3.4 1/12/98 (4° C., Air) 1.53.8 3.9 3.4 1/12/98 (R.T., N₂) 0.1 1.4 0.7 0.7 1/12/98 (R.T., Air) 0.41.6 1.0 0.4 4/30/98 (4° C., N₂) 0.08 4.3 4.0 5.3 4/30/98 (4° C., Air)1.5 3.6 4.4 4.5 4/30/98 (R.T., N₂) 0.0025 0.23 0.11 0.17 4/30/98 (R.T.,Air) 0.0015 0.21 0.063 0.007 2/5/99 (4° C., N₂) 0.0005 5.8 4.1 3.92/5/99 (4° C., Air) 0.02 4.7 4.3 4.5 2/5/99 (R.T., N₂) <10² 0.0007 <10⁴0.0002 2/5/99 (R.T., Air) 2 × 10² 0.0002 0.0003 2 × 10³ Date HPLC ViralParticle (×10¹⁰/ml) (Storage Conds.) 10%-G 5%-S + 5%-HSA 5%-S + 1%-PEG5%-T + 1%-PEG 8/1/97 16.9 14.5 16.1 16.7 8/28/97 (4° C., N₂) 13.3 14.913.8 13.4 8/28/97 (4° C., Air) 12.9 14.2 12.9 12.9 8/28/97 (R.T., N₂)12.6 14.5 13.5 12.9 8/28/97 (R.T., Air) 12.3 13.7 13.0 13.0 10/30/97 (4°C., N₂) 14.0 15.5 14.7 14.8 10/30/97 (4° C., Air) 12.6 14.9 14.3 14.410/30/97 (R.T., N₂) 13.8 15.1 14.6 14.4 10/30/97 (R.T., Air) 12.7 14.714.8 14.4 1/12/98 (4° C., N₂) 7.3 11.1 9.5 9.5 1/12/98 (4° C., Air) 7.710.8 10.2 10.0 1/12/98 (R.T., N₂) 10.0 10.8 11.1 10.4 1/12/98 (R.T.,Air) 9.9 11.0 10.0 10.4 4/30/98 (4° C., N₂) 5.1 12.3 12.3 12.1 4/30/98(4° C., Air) 5.0 11.6 11.8 11.9 4/30/98 (R.T., N₂) 11.1 12.3 12.6 12.54/30.98 (R.T., Air) 11.0 12.4 12.3 11.0 2/5/99 (4° C., N₂) 3.4 5.8 11.411.0 2/5/99 (4° C., Air) 3.9 7.1 11.0 11.2 2/5/99 (R.T., N₂) 10.1 7.98.5 10.9 2/5/99 (R.T., Air) 9.7 7.1 10.3 9.3

[0232] TABLE 13 Aqueous Formulation Set #2 PFU × 10⁹/ml Date (Temp.)AQF2-1 AQF2-2 AQF2-3 AQF2-4 AQE2-5 AQF2-6 9/25/97 2.8 2.8 2.8 3.0 2.82.8 11/05/97 2.3 3.2 2.4 3.6 2.7 2.0 (4° C.) 11/05/97 1.4 1.9 1.3 1.52.4 2.5 (R.T.) 12/12/97 2.2 0.1 2.4 2.7 2.1 2.1 (4° C.) 1/09/98 1.2 0.10.2 1.2 0.2 0.1 (R.T.) PFU × 10⁹/ml AQF2- AQF2- AQF2- AQF2- AQF2- AQF2-Date (Temp.) 7* 8* 9* 10* 11* 12 9/25/97 2.7 2.8 2.7 3.3 3.1 2.711/05/97 3.6 3.8 2.7 3.0 3.5 2.5 (4° C.) 11/05/97 3.1 3.3 3.1 4.1 2.81.1 (R.T.) 12/12/97 3.2 2.1 3.0 3.0 3.4 2.9 (4° C.) 1/09/98 1.3 1.1 0.20.1 20 1.1 (R.T.) HPLC viral particle (× 10¹⁰/ml) Date (Temp.) AQF2-1AQE2-2 AQF2-3 AQF2-4 AQF2-5 AQF2-6 9/25/97 10.9 9.6 9.7 11.3 10.7 10.611/05/97 7.9 7.6 8.7 8.8 8.9 7.5 (4° C.) 11/05/97 8.2 6.6 7.6 8.6 7.79.3 (R.T.) 12/12/97 6.7 1.5 8.0 6.9 5.2 7.5 (4° C.) 12/17/97 7.0 1.2 7.07.5 4.1 7.1 (R.T.) HPLC viral particle (× 10¹⁰/ml) AQF2- AQF2- AQF2-Date (Temp.) AQF2-7 AQF2-8 AQF2-9 10 11 12 9/25/97 10.9 10.8 10.7 11.411.8 10.7 11/05/97 8.6 9.1 9.2 10.3 11.2 9.6 (4° C.) 11/05/97 9.0 8.09.3 10.3 11.1 9.6 (R.T.) 12/12/97 7.5 6.1 7.6 8.8 7.3 7.7 (4° C.)12/17/97 7.0 3.0 8.2 7.6 8.4 7.5 (R.T.) Excipients AQF2-1 AQF2-2 AQF2-3AQF2-4 AQF2-5 AQF2-6 mannitol 5 5 5 (W %) sucrose (W %) 5 5 5 glycine(M) 0.25 0.25 arginine (M) 0.25 0.25 urea (W %) 1 1 peg (w %) AQF2-AQF2- AQF2- Excipients AQF2-7 AQF2-8 AQF2-9 10 11 12 mannitol 5 5 5 5 5(W %) sucrose (W %) 5 5 5 5 5 10 glycine (M) 0.25 0.25 0.25 arginine (M)0.25 0.25 urea (W %) 1 1 peg (w %) 1 1

[0233] TABLE 14 Aqueous Formulation Set #3 HPLC Viral PFU × 10⁹ Particle(× 10⁹/ml) F10-7 F10-8 F11-7 F11-8 F10-7 F10-8 F11-7 F11-8 Date (temp.)10/3/97 2.2 3.3 2.1 2.8 12.1 12.0 11.8 12.0 11/6/97 3.4 4.0 2.8 3.4 10.610.5 10.1 10.3 (−20° C.) 11/6/97 (4° C.) 3.5 3.6 4.3 2.8 10.0 9.7 9.910.3 1/15/98 3.8 4.8 3.2 3.7 7.3 7.4 7.7 8.0 (−20° C.) 1/15/98 (4° C.)3.5 3.1 2.9 3.1 7.5 7.4 7.6 7.5 Excipients mannitol 6 6 5 5 (W %)sucrose (W %) 7 8 7 8 HSA (W %) 0.5 0.5 0.5 0.5 glycerol (W %) 1 1 1 1MgCl₂ (mM) 1 1 1 1

[0234] TABLE 15 Liquid formulation set #4 AQF4-1 AQF4-2 AQF4-3 AQF4-4AQF4-5 AQF4-6 AQF4-7 Date (Temp.) PFU (× 10⁹/ml) 1/13/98 3.0 2.5 3.6 3.42.7 3.1 3.4 2/16/98 (4° C.) 2.5 3.2 3.3 2.9 2.6 2.9 2.6 2/16/98 (R.T.)1.8 2.7 1.6 3.6 2.6 1.6 1.7 4/10/98 (4° C.) 2.2 2.0 2.6 3.0 2.4 1.9 2.24/10.98 (R.T.) 0.4 0.4 0.3 0.5 0.4 <0.1 1.1 7/24/98 (4° C.) 2.4 2.8 2.63.5 1.9 2.2 2.6 7/24/98 (R.T.) 0.002 0.005 0.006 0.005 0.005 0.005 0.0011/8/99 (4° C.) 2.9 2.4 2.1 2.6 2.0 2.2 2.1 1/8/99 (R.T.) 0.0002 0.00040.0004 0.0002 0.0004 0.0004 0.00006 Date (Temp.) HPLC Viral Particles (×10¹⁰/ml) 1/13/98 7.2 8.8 9.2 9.0 7.8 7.9 9.1 2/16/98 (4° C.) 7.5 9.3 9.29.5 8.2 8.4 9.6 2/16/98 (R.T.) 6.8 9.0 9.5 9.0 8.7 8.4 9.3 4/10/98 (4°C.) 7.1 9.2 9.6 9.6 8.9 9.1 9.9 4/10/98 (R.T.) 7.5 9.5 10.1 9.7 8.9 8.99.5 7/24/98 (4° C.) 8.1 9.9 11.1 10.3 9.2 7.4 9.3 7/24/98 (R.T.) 7.3 3.010.7 8.9 10.4 10.45 3.5 1/8/99 (4° C.) 7.8 10.3 10.3 10.1 8.7 1.7 9.51/8/99 (R.T.) 8.4 11.0 11.3 11.0 9.7 10.4 9.4 Excipients Mannitol (W %)5 5 5 5 5 5 5 Sucrose (w %) 5 5 5 5 5 5 5 Tween −80 (w %) 0.02 0.1 0.5Chap (w %) 0.02 0.1 0.5

[0235] Table 10 and Table 11 show the storage stability data withsecondary drying at 30° C. without and with N₂ backfilling,respectively. Because of the nearly identical stability observed at −20°C. and 4° C. storage conditions, and to reduce the consumption of virus,−20° C. was not included in the long-term storage stability study.Similar to the samples dried with secondary drying at 10° C., virus isstable at 4° C. but not stable at RT. However, relative better stabilitywas observed at RT storage than those dried at 10° C. secondary drying.This is likely to be the result of the lower residual moisture attainedat 30° C. secondary drying. This result suggests that residual moistureis an important parameter that affects storage stability during longterm storage.

[0236] HPLC viral particle recoveries are consistently lower than virusrecoveries calculated from PFU assay immediate after drying. The reasonfor the discrepancy is not clear. However, it is likely to be related topossible virus aggregation during freeze-drying. Electron microscopyevaluation is being carried out to examine possible virus aggregationafter lyophilization. During storage, HPLC analysis indicates that virusis stable at both −20° C. and 4° C. storage and not stable at RT, whichis consistent with the results from PFU assay.

Example 5 HSA Alternatives

[0237] The presence of HSA in the formulations could be a potentialregulatory concern. As a result, a variety of excipients have beenevaluated to substitute HSA in the formulation. The substitutes examinedincluded PEG, amino acids (glycine, arginine), polymers(polyvinylpyrrolidone), and surfactants (Tween-20 and Tween-80). TheseHSA substitutes are, however, suboptimal relative to HSA. Effort onfurther development was minimal.

Example 6 Liquid Formulation

[0238] Concurrent with the development of lyophilization of Adp53product, experimentation was carried out to examine the possibility ofdeveloping a liquid formulation for Adp53 product. The goal was todevelop a formulation that can provide enough stability to the viruswhen stored at above freezing temperatures. Four sets of liquidformulations have been evaluated. In the first set of formulation, thecurrent 10% glycerol formulation was compared to HSA and PEG containingformulations. In the second set of formulation, various amino acids wereexamined for formulating Adp53. In the third set of formulation, theoptimal formulation developed for lyophilization was used to formulateAdp53 in a liquid form. In the fourth set of formulation, detergentswere evaluated for formulating Adp53. Viruses formulated with all thosedifferent formulations are being tested for long term storage stabilityat −20° C., 4° C., and RT.

[0239] Liquid Formulation Set #1

[0240] HSA containing formulation (5% sucrose+ 5% HSA in 10 mM Trisbuffer, 150 mM NaCl, and 1 mM MgCl₂, pH=8.20 buffer) was compared with10% glycerol in DPBS buffer and sucrose/PEG and Trehalose/PEGformulations. PEG has been recommended as a good preferential exclusionagent in formulations (Wong and Parasrampurita, 1997). It is included inthis set of formulation to examine whether it can provide stabilizationeffect on Adp53. Formulations were filled into the 3 ml lyo vials at afill volume of 0.5 ml. Vials were capped under either atmospheric or N₂blanketing conditions to examine any positive effects N₂ blanketing mayhave on long term storage stability of Adp53. To ensure adequatedegassing from the formulation and subsequent N₂ blanketing, the filledvials was partially stoppered with lyo stoppers and loaded onto theshelf of the lyophilizer under RT. The lyophilizer chamber was closedand vacuum was established by turning on the vacuum pump. The chamberwas evacuated to 25 in Hg. Then the chamber was purged completely withdry N₂. The evacuation and gassing were repeated twice to ensurecomplete N₂ blanketing. N₂ blanketed vials were placed with the non-N₂blanketed vials at various storage conditions for storage stabilityevaluation. Table 12 shows the analysis data for up to 18 months storageat 4° C. and RT.

[0241] Statistically significant drops in virus PFU and HPLC viralparticles were observed for 10% glycerol formulation after 3 monthsstorage at both 4° C. and RT. No statistically significant virusdegradation was observed for all other formulations at 4° C. storage.However, decrease in virus infectivity was observed when stored at RT.

[0242] Liquid Formulation Set #2

[0243] Various combinations of amino acids, sugars, PEG and urea wereevaluated for Adp53 stabilization during long storage. Table 13 showsthe 12-month stability data. The results indicate that combination of 5%mannitol and 5% sucrose with other excipients gave better storagestability at RT for one month. Adp53 is most stable in formulation hasall the excipients. In this set of formulation, no human or animalderived excipients were included. It is our expectation to develop aliquid formulation without including any proteins derived from eitherhuman or animal origins.

[0244] Liquid Formulation Set #3

[0245] The optimal formulations developed for lyophilization wasevaluated for formulating Adp53 in a liquid form. This approach would bea good bridging between liquid formulation and lyophilization ifsatisfactory Adp53 stability can be achieved using lyophilizationformulation for liquid fill. Filled samples were stored at −20° C. and4° C. for stability study. Table 14 shows the 3-month stability data.Virus is stable at both −20° C. and 4° C. for the four differentformulations. This is in agreement with the results from formulation set#2, which suggests that better virus stability is expected with thepresence of both mannitol and sucrose in the formulation. Longer timestorage stability data is being accrued.

[0246] Liquid Formulation Set #4

[0247] Detergents have been used in the formulations for a variety ofrecombinant proteins. In this set of formulation, various concentrationsof detergents were examined for formulating Adp53. The detergents usedwere non-ionic (Tween-80) and zwitterionic (Chap). Table 15 shows the12-month stability data. Virus is stable at 4° C. storage. Nosignificant difference in virus stability at 4° C. was observed amongthe formulations tested. Similar to formulation set #2, no exogenousprotein is included in this set of formulation.

[0248] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A pharmaceutical adenovirus compositioncomprising adenovirus particles and pharmaceutical excipients, theexcipients including a bulking agent and one or more protectants,wherein the excipients are included in amounts effective to provide anadenovirus composition that is storage stable.
 2. The adenoviruscomposition of claim 1, further defined as having an infectivity ofbetween 60 and 100% of the starting infectivity, and a residual moistureof less than about 5%, when stored for six months at 4° centigrade. 3.The adenovirus composition of claim 1, further defined as a freeze driedcomposition.
 4. The composition of claim 3, wherein the bulking agent isfurther defined as a bulking agent which forms crystals during freezing.5. The composition of claim 1, wherein the bulking agent is mannitol,inositol, lactitol, xylitol, isomaltol, sorbitol, gelatin, agar, pectin,casein, dried skim milk, dried whole milk, silcate,carboxypolymethylene, hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methhylcellulose or methylcellulose.
 6. The composition ofclaim 5, wherein said bulking agent is mannitol.
 7. The composition ofclaim 6, further defined as an aqueous composition comprising thebulking agent in a concentration of from about 1% to about 10% (w/v). 8.The composition of claim 7, wherein the aqueous composition comprisesthe bulking agent in a concentration of from about 3% to 8%.
 9. Thecomposition of claim 8, wherein the aqueous composition comprises thebulking agent in a concentration of from about 5% to 7%.
 10. Thecomposition of claim 3, wherein the freeze dried composition wasprepared from an aqueous composition comprising a bulking agent in aconcentration of from about 1% to 10% (w/v).
 11. The composition ofclaim 10, wherein the freeze dried composition was prepared from anaqueous composition comprising a bulking agent in a concentration offrom about 3% to 8%.
 12. The composition of claim 11, wherein the freezedried composition was prepared from an aqueous composition comprising abulking agent in a concentration of from about 5% to 7%.
 13. Thecomposition of claim 1, wherein said protectant is further defined asincluding a cryoprotectant.
 14. The composition of claim 13, whereinsaid cryoprotectant is a non-reducing sugar.
 15. The composition ofclaim 14, wherein the non-reducing sugar is sucrose or trehalose. 16.The composition of claim 15, wherein said cryoprotectant is sucrose. 17.The composition of claim 14, further defined as an aqueous compositioncomprising the non-reducing sugar in a concentration of from about 2% toabout 10% (w/v).
 18. The composition of claim 17, wherein the aqueouscomposition comprises the sugar in a concentration of from about 4% to8%.
 19. The composition of claim 18, wherein the aqueous compositioncomprises the sugar in a concentration of from about 5% to 6%.
 20. Thecomposition of claim 3, wherein the freeze dried composition wasprepared from an aqueous composition comprising a non-reducing sugar ina concentration of from about 2% to 10% (w/v).
 21. The composition ofclaim 20, wherein the freeze dried composition was prepared from anaqueous composition comprising a non-reducing sugar in a concentrationof from about 4% to 8%.
 22. The composition of claim 21, wherein thefreeze dried composition was prepared from an aqueous compositioncomprising a non-reducing sugar in a concentration of from about 5% to6%.
 23. The composition of claim 13, wherein the cryoprotectant isniacinamide, creatinine, monosodium glutamate, dimethyl sulfoxide orsweet whey solids.
 24. The composition of claim 1, wherein saidprotectant includes a lyoprotectant.
 25. The composition of claim 24,wherein said lyoprotectant is human serum albumin, bovine serum albumin,PEG, glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80.
 26. The composition of claim 25, wherein said lyoprotectantis human serum albumin.
 27. The composition of claim 24, further definedas an aqueous composition comprising the lyoprotectant in aconcentration of from about 0.5% to about 5% (w/v).
 28. The compositionof claim 27, wherein the aqueous composition comprises the lyoprotectantin a concentration of from about 1% to about 4%.
 29. The composition ofclaim 28, wherein the aqueous composition comprises the lyoprotectant ina concentration of from about 1% to about 3%.
 30. The composition ofclaim 3, wherein the freeze dried composition was prepared from anaqueous composition comprising a lyoprotectant in a concentration offrom about 0.5% to 5% (w/v).
 31. The composition of claim 30, whereinthe freeze dried composition was prepared from an aqueous compositioncomprising a lyoprotectant in a concentration of from about 1% to 4%.32. The composition of claim 31, wherein the freeze dried compositionwas prepared from an aqueous composition comprising a lyoprotectant in aconcentration of from about 1% to 3%.
 33. The composition of claim 24,further defined as comprising both a lyoprotectant and a cryoprotectant.34. An aqueous pharmaceutical adenovirus composition comprising a polyolin an amount effective to promote the maintenance of adenoviralinfectivity.
 35. The composition of claim 34, further defined asmaintaining an infectivity of about 70% PFU/mL to about 99.9% PFU/mL ofthe starting infectivity when stored for six months at 4° centigrade.36. The composition of claim 34, further defined as maintaining aninfectivity of about 80% to 95% PFU/mL of the starting infectivity whenstored for six months at 4° centigrade.
 37. The composition of claim 34,wherein said polyol is glycerol,- propylene glycol, polyethylene glycol,sorbitol or mannitol.
 38. The composition of claim 34, wherein saidpolyol concentration is from about 5% to about 30% (w/v).
 39. Thecomposition of claim 38, wherein said polyol concentration is from about10% to about 30%.
 40. The composition of claim 34, wherein said polyolis glycerol, included in a concentration of from about 10% to about 30%(w/v).
 41. The composition of claim 34, wherein said composition furthercomprises an excipient in addition to said polyol, wherein saidexcipient is inositol, lactitol, xylitol, isomaltol, gelatin, agar,pectin, casein, dried skim milk, dried whole milk, silicate,carboxypolymethylene, hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methhylcellulose, methylcellulose, sucrose, dextrose,lactose, trehalose, glucose, maltose, niacinamide, creatinine,monosodium glutamate dimethyl sulfoxide, sweet whey solids, human serumalbumin, bovine serum albumin, PEG, glycine, arginine, proline, lysine,alanine, polyvinyl pyrrolidine, polyvinyl alcohol, polydextran,maltodextrins, hydroxypropyl-beta-cyclodextrin, partially hydrolysedstarches, Tween-20 or Tween-80.
 42. The composition of claim 41, whereinsaid composition further comprises at least a first and second of saidexcipients, said second excipient different from said first excipient.43. A method for preparation of a long-term, storage stable adenovirusformulation, comprising the steps of: (a) providing adenovirus andcombining said adenovirus with a solution comprising a buffer, a bulkingagent, a cryoprotectant and a lyoprotectant; and (b) lyophilizing saidsolution, whereby lyophilization of said solution produces afreeze-dried cake of said adenovirus formulation that retains highinfectivity and low residual moisture.
 44. The method of claim 43,wherein said bulking agent is mannitol, inositol, lactitol, xylitol,isomaltol, sorbitol, gelatin, agar, pectin, casein, dried skim milk,dried whole milk, silcate, carboxypolymethylene, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methhylcellulose ormethylcellulose.
 45. The method of claim 44, wherein said bulking agentis mannitol.
 46. The method of claim 45, wherein mannitol comprisesabout 0.5% to about 8% (w/v) of said formulation.
 47. The method ofclaim 43, wherein said cryoprotectant is sucrose, dextrose, lactose,trehalose, glucose, maltose, niacinamide, creatinine, monosodiumglutamate dimethyl sulfoxide or sweet whey solids.
 48. The method ofclaim 47, wherein said cryoprotectant is sucrose.
 49. The method ofclaim 43, wherein said sucrose comprises about 2.5% to about 10% (w/v)of said formulation.
 50. The method of claim 43, wherein saidIyoprotectant is human serum albumin, bovine serum albumin, PEG,glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80.
 51. The method of claim 50, wherein said lyoprotectant ishuman serum albumin.
 52. The method of claim 43, wherein said buffer isTris-HCl, TES, HEPES, mono-Tris, brucine tetrahydrate, EPPS, tricine, orhistidine.
 53. The method of claim 52, wherein said buffer is present insaid formulation at a concentration at about 1 mM to 50 mM.
 54. Themethod of claim 53, wherein said buffer is Tris-HCl.
 55. The method ofclaim 54, wherein said Tris-HCl is included in a concentration of fromabout 1 mM to about 50 mM.
 56. The method of claim 55, wherein saidTris-HCl is included in a concentration of from about 5 mM to about 20mM.
 57. The method of claim 43, further comprising a salt selected fromthe group consisting of MgCl₂, MnCl₂, CaCl₂, ZnCl₂, NaCl and KCl. 58.The method of claim 43, wherein said lyophilizing is carried out in thepresence of an inert gas.
 59. The method of claim 43, whereinlyophilizing said solution comprises the steps of: (a) freezing saidsolution; (b) subjecting said solution to a vacuum; and (c) subjectingsaid solution to at least a first and a second drying cycle, wherebysaid second drying cycle reduces the residual moisture content of saidfreeze-dried cake to less than about 2%.
 60. A method for thepreparation of a long-term storage, stable adenovirus liquidformulation, comprising the steps of providing adenovirus and combiningsaid adenovirus with a solution comprising a buffer and a polyol,whereby said adenovirus liquid formulation retains high infectivity.